Datasets:

minh21

/

COVID-QA-question-answering-biencoder-data-45_45_10

like 0

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: i the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, ii the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, iii the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and iv the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use.", "Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage genera Inovirus and Plectrovirus are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb .", "Subsequent to the independent discovery of bacteriophage by Twort . and d 'Hérelle . , the first filamentous phage, f1, was isolated in Loeb . and later characterized as a member of a larger group of phage Ff, including f1, M13, and fd phage specific for the E. coli conjugative F pilus Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964 . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species.", "Soon thereafter, filamentous phage were discovered that do not use F-pili for entry If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972 , and over time the list of known filamentous phage has expanded to over 60 members . , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012 . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues Smith, 1985; Parmley and Smith, 1988 . Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 .", "Based on the ideas described in Parmley and Smith . , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991 . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010 . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications.", "However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: i filamentous phage as a vaccine carrier; ii engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; iii filamentous phage as a scaffold for bioconjugation and surface chemistry; and iv filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation.", "A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins Table 1 . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage.", "Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome type 8 display on pVIII, type 3 display on pIII, etc. , resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith ., McConnell et al. . , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott .", ". , Rondot et al. . Hybrid type 33 and 3+3 systems Type 3+3 system <1 2 Smith and Scott . , Smith and Petrenko . pVI Hybrid type 6+6 system Yes <1 2 >25 kDa Hufton et al. . pVII Fully recombinant type 7 system No ∼5 >25 kDa Kwasnikowski et al. . Hybrid type 7+7 system Yes <1 2 Gao et al. . pVIII Fully recombinant landscape phage; type 8 system No 2700 3 ∼5-8 residues Kishchenko et al. . , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. .", ". , Petrenko et al. . Hybrid type 88 and 8+8 systems Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith . , Greenwood et al. . , Smith and Fernandez . pIX Fully recombinant type 9+9 * system Yes ∼5 >25 kDa Gao et al. . Hybrid type 9+9 system No <1 2 Gao et al. . , Shi et al. . , Tornetta et al. . 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide.", ". 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display .", "recombinant gene 8 is on a plasmid with a phage origin of replication resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene e.g., type 3 * +3 display . By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions , and of the latter being the ability to display some folded proteins at an appreciable copy number 1-5 per phage particle . While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion . . For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides .", ". For the purposes of this review, we use the term \"phage display\" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins , and the term \"phage-displayed library\" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants e.g., antibody fragments; peptides . Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants Meynell and Lawn, 1968 and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies Pratt et al., 1969; Woolford et al., 1977 . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. .", "Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. . . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen via pVIII display also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . .", "Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII . . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009 were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens or peptide ligands of antibodies against these antigens; Table 2 , including malaria and human immunodeficiency virus type 1 HIV-1 . When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 .", "When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996 , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants or mimics thereof of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses di Marzo Veronese et al., 1994; Scala et al., 1999 . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens Table 2 . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: i in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins Irving et al., 2010 ; see below ; ii antibodies against a single epitope may be of limited utility, especially for highly variable pathogens Van Regenmortel, 2012 ; and iii for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease .", "More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs Frenkel et al., 2000 Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011 , possibly reduced amyloid plaque formation in mice Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008 , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease . ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation.", "Yip et al. . found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules . , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes . . Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development.", "Phage displaying or chemically Rubinchik and Chow . conjugated to sperm antigen peptides or peptide mimics Samoylova et al., 2012a,b and gonadotropin-releasing hormone . are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals Table 2 , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions . possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target .", "possibly due to copy number differences pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996 . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin BSA and keyhole limpet hemocyanin KLH; Melzer et al., 2003; Su et al., 2007 , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target . . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al.", "The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: i peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or ii the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. . describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins .", "While the peptide may mimic the chemistry of a given epitope on a folded protein allowing it to crossreact with a targeted antibody , being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins . . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity see Immunological Mechanisms of Vaccination with Filamentous Phage below . The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 .", "The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII Table 3 . Early work, showing that immunization with phage elicited T-cell help Kölsch et al., 1971; Willis et al., 1993 , was confirmed by several subsequent studies De Berardinis et al., 1999; Ulivieri et al., 2008 . From the perspective of vaccination against infectious disease, De Berardinis et al. . showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . .", "showed that a cytotoxic T-cell CTL epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins . . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus . and Candida albicans Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation.", "Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial Manoutcharian et al., 2004; Morales et al., 2008 . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies Plotkin, 2010 , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production . . E. coli F17a-G adhesin . , hepatitis B core antigen .", ". E. coli F17a-G adhesin . , hepatitis B core antigen . , and hepatitis B surface antigen . all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone . . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . .", "Immunization with phage displaying the 1E10 idiotype scFv mimicking a Vibrio anguillarum surface epitope elicited antibodies that protected flounder fish from Vibrio anguillarum challenge . . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model . , and was welltolerated and immunogenic in patients with multiple myeloma . . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins.", "One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone . , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice .", "Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination Figure 1 . The phage particle is immunogenic without adjuvant in all species tested to date, including mice . , rats . , rabbits . , guinea pigs Frenkel et al., 2000; Kim et al., 2004 , fish Coull et al., 1996; Xia et al., 2005 , non-human primates . , and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal .", ", and humans . . Various routes of immunization have been employed, including oral administration . as well as subcutaneous . , intraperitoneal . , intramuscular Samoylova et al., 2012a , intravenous Vaks and Benhar, 2011 , and intradermal injection . ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: i the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice Terry et al., 1997; Kneissel et al., 1999 ; ii the N-terminal N1 and N2 domains of pIII . ; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . .", "; and iii bacterial lipopolysaccharide LPS embedded in the phage coat . . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization . . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes . . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . .", "Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity . . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity macrophages, neutrophils, and possibly natural killer cells , which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses.", "The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | Although serum anti-phage antibody titers appear to be at least partially T-cell dependent Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010 , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses Murira, 2014 . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules Gaubin et al., 2003; Ulivieri et al., 2008 and can activate T H 1, T H 2, and T H 17 helper T cells Yang et al., 2005a; Wang et al., 2014d . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . .", "Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII . . Phage proteins can also be cross-presented on MHC class I molecules . and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help . . The latter CTLs mediate a delayed-type hypersensitivity reaction Fang et al., 2005; Del Pozzo et al., 2010 . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . .", "Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins . . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors . , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days .", "Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system . , particularly of the liver and spleen, where it is retained for days . , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . .", "Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe . . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a \"Trojan horse\" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 .", "M13 and Pf3 phage engineered to express either BglII restriction endonuclease Hagens and Blasi, 2003; Hagens et al., 2004 , lambda phage S holin Hagens and Blasi, 2003 or a lethal catabolite gene activator protein . effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS Hagens and Blasi, 2003; Hagens et al., 2004 . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 .", "Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage . or phage engineered to repress the cellular SOS response Lu and Collins, 2009 . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli . . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 .", "Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity Figure 2 . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression Poul and Marks, 1999 . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol Yacoby et al., 2006; Vaks and Benhar, 2011 . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c .", "M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro Ghosh et al., 2012a . Tumorspecific peptide:pVIII fusion proteins selected from \"landscape\" phage Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models Jayanna et al., 2010b; Wang et al., 2014b,c . Using the B16-OVA tumor model, Eriksson et al. . showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . .", "showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site . . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic Frenkel and Solomon, 2002 and therapeutic Solomon, 2008 reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue . . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . .", "Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis . . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are i its ability to carry very high amounts of drug or peptide, and ii its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography .", "First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units EU /mL Boratynski et al., 2004; Branston et al., 2015 , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation Smith and Gingrich, 2005; Branston et al., 2015 , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography Boratynski et al., 2004; Zakharova et al., 2005 , polymyxin B chromatography . , and treatment with detergents such as Triton X-100 or Triton X-114 Roehnisch et al., 2014; Branston et al., 2015 . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate LAL assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline .", "A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline . or for detection of foodborne pathogens post-production reviewed in Schmelcher and Loessner, 2014 . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance .", "M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance . , piezoelectric transducers . , linear dichroism Pacheco-Gomez et al., 2012 , and magnetoelastic sensor technology Lakshmanan et al., 2007; Huang et al., 2009 were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . .", "Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce Li et al., 2010b and eggs . . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups . . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules.", "The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation Figure 3 . The most commonly used approach is functionalization of amine groups with NHS esters van Houten et al., 2006 van Houten et al., , 2010 Yacoby et al., 2006 , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively Li et al., 2010a . Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction.", "Carrico et al. . developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides . or enzymes Chen et al., 2007; Hess et al., 2012 , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . .", "It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions . . Lee et al. . engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice.", "Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012 , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups shown in red , three carboxyl groups show in blue and two hydroxyl groups show in green . The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 .", "Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires Mao et al., 2003 Mao et al., , 2004 , nanoparticles , and nanocomposites Oh et al., 2012; Chen et al., 2014 . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. . produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . .", ". produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors . . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides . , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging Ghosh et al., 2012b; Yi et al., 2012 , photocatalytic water splitting Nam et al., 2010a; Neltner et al., 2010 , light harvesting Nam et al., 2010b; Chen et al., 2013 , photoresponsive technologies . , neural electrodes . , and piezoelectric energy generation . .", ", neural electrodes . , and piezoelectric energy generation . . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy.", "It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions Hansen et al., 1998 Hansen et al., , 2000 Dahlke Ojennus et al., 1999 in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013 . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 .", "First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system Marks et al., 1992; Bradbury et al., 2011 . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation although each display technology has complementary strengths; Koide and Koide, 2012 , and regardless of the display method, selection of \"improved\" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations . and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al.", "and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. . developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution PACE , which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al.", "The authors engineered M13 phage to encode an exogenous protein the subject for directed evolution , whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. . elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. .", ". elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. . later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution .", "PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis Meyer and Ellington, 2011 . This approach represents a promising avenue for both basic research in molecular evolution . and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil . , coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . .", ", coastal fresh water . , alpine lakes Hofer and Sommaruga, 2001 and deep sea bacteria . , but not, perhaps surprisingly, the human gut . . The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 .", "The environmental \"phageome\" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005 . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques typically direct observation by electron microscopy found that filamentous phage made up anywhere from 0 to 100% of all viral particles Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001 . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria Hofer and Sommaruga, 2001 . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water .", "The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater . and reclaimed and potable water . but have much higher frequencies in wastewater and sewage Cantalupo et al., 2011; Alhamlan et al., 2013 , with the caveat that biases inherent to the methodologies for ascertaining these data purification of viral particles, sequencing biases have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage.", "At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution . . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 .", "Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage Waldor and Mekalanos, 1996 . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 . . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus .", "Infection by filamentous phage has been implicated in the virulence of Yersinia pestis . , Neisseria meningitidis Bille et al., 2005 Bille et al., , 2008 , Vibrio parahaemolyticus . , E. coli 018:K1:H7 . , Xanthomonas campestris Kamiunten and Wakimoto, 1982 , and P. aeruginosa . , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 .", ", although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum Yamada, 2013 . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes . . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 .", "Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants Petrenko and Makowski, 1993 , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings Curtis et al., 2011 Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies Curtis et al., 2009 . The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials.", "Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript.", "While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text." ]

[ "Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths 19.2 to 103.5X after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages.", "Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding 10 6.42 –10 7.01 copies/swab and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread n = 25, 56.8% positive of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted.", "MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded. Text: metagenomics is a valuable asset for diagnostics in pigs, leading to discovery of novel viruses and identification of porcine viral enteric disease complexes. Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective.", "Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective. Performing the necessary analyses often results in long time periods between sample arrival and diagnostic reporting, since results can only be processed after finishing the sequencing run. Third-generation sequencing using MinION Oxford Nanopore Technologies, ONT might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides.", "The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides. This signal is converted into a nucleotide sequence by computational algorithms basecalling . Since the release of MinION technology, major advances have been made in terms of the number and the quality of reads generated 15 . In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 .", "In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 . MinION has also been used as a diagnostic tool during recent Ebolavirus outbreaks in West Africa, allowing fast on-site characterization of circulating strains 17, 18 . Coupled to a laptop-based bioinformatics workflow, MinION was able to detect Chikungunya virus, Ebola virus and hepatitis C virus in less than 6 hours using earlier versions of the technology 19 . A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 .", "A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 . In veterinary virology, the use of nanopore sequencing is growing. A novel species of papillomavirus was identified in warts from giraffes, using rolling-circle amplification and nanopore sequencing 22 . The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 .", "The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 . To the author's knowledge, the present study is the first using MinION as an aid in porcine health management. This study was aimed to explore the possibilities of MinION as a rapid and easy-to-use diagnostic tool in pig health management for diagnosis of viral enteric disease complexes. The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated.", "The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated. No gene-specific or random pre-amplification of viral nucleic acids was conducted to challenge the MinION's sensitivity. A porcine kobuvirus was discovered in the latter case and a longitudinal field study was conducted hereafter to elucidate the shedding patterns of this virus. Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium.", "Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium. be performed for 243,313 reads with a mean length of 740 nucleotides. Reads with a q-score lower than 7 were filtered out, resulting in 179,015 remaining sequences mean length 816 nt for use in downstream analyses. Results of the sequencing run, including taxonomical classification and mapping of reads against PEDV and rotavirus A RVA reference genomes are shown in Fig. 1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database.", "1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database. Of these, 39.3% n = 5,985 and 10.3% n = 1,564 were assigned to viral families comprising Porcine epidemic diarrhea virus family Coronaviridae and Rotavirus family Reoviridae, subfamily Sedoreovirinae , respectively. A fraction of the reads 29.3%, n = 4,468 were assigned to order Caudovirales. These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore.", "These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore. Most reads were generated in the first twelve hours of sequencing and read accumulation was most exponential in the first three hours of sequencing Fig. 1B . PEDV and RVA sequences were extracted from the dataset and mapped against viral reference genes to calculate sequencing depths over time Fig. 1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X .", "1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X . High sequencing depths were acquired after three hours of sequencing for PEDV 103.5X and for most RVA gene segments 19.2 to 48.2X . De novo assembly was executed on the quality-filtered reads prior to identification tBLASTx to recover viral genomes. This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 .", "This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 . However, execution of de novo assembly prior to taxonomical classification tBLASTx reduced the time to identify entire viral genomes in the dataset. Virome composition of a young diarrheic piglet using nanopore sequencing. A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses.", "A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses. Different methods were used to compare the reads against a viral database using the HPC cluster of Ghent University and results are shown in Fig. 2 . Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx.", "Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx. However, there was a very high difference between wall times on the HPC cluster, with only 26 seconds of analysis time for BLASTn, versus almost 24 hours for tBLASTx. The majority of sequences were assigned to bacteriophages within the order Caudovirales and families Siphoviridae n = 3,213 to 4,163 reads , Podoviridae n = 2,506 to 3,002 reads and Myoviridae n = 912 to 1,202 reads . A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig.", "A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig. 2B . Comparison of these contigs against the GenBank database using BLAST allowed classification into four different groups. Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown .", "Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown . The longest contig with a size of 39,069 nucleotides, together with four other contigs showed similarities 95% nt identity to different Escherichia phages. As they also mapped nicely distributed across the reference genome of Escherichia phage vB_EcoP_PhAPEC7, it seems that they must also belong to one phage genome data not shown . Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome.", "Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome. Interestingly, three contigs were identified for which no similarities were found with existing viruses in GenBank, but contig 0105 mapped to the reference genome of the Enterococcus phage vB_EfaS_IME196 data not shown . These might be novel phages or divergent variants from existing phages present in GenBank. Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae.", "Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae. Kobuvirus reads were mapped against a European reference strain S-1/HUN/2007/Hungary, as shown in Fig. 2C . However, full-genome coverage at high sequencing depth was not obtained. Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated.", "Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated. The fecal shedding patterns of the different viruses and presence of diarrheic signs are shown in Fig. 3A . All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification .", "All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification . Peak shedding titers of the porcine kobuvirus varied between 6.42 and 7.01 log 10 copies/swab, which is generally lower than peak shedding observed for typical enteric viruses such as rotavirus and PEDV. Moreover, the peak of shedding was not related to diarrheic episodes, questioning the role of this virus in the pathogenesis of diarrhea on the farm. Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed.", "Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed. In contrast, there was no direct association between peak shedding of kobuvirus and diarrheic episodes. Interestingly, a peak in kobuvirus shedding was observed in piglet C at day 11 post-farrowing. This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine.", "This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine. Retrospective analysis of porcine kobuviruses shedding in Belgian diarrheic suckling pigs and phylogenetic analysis. A total of 44 diarrheic fecal samples collected in 2014 were screened for the presence of kobuvirus using the new RT-qPCR. Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification.", "Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification. The presence of RVA and RVC had been quantitatively assessed in these samples in a previous study and the occurrence of co-infections between rotaviruses and kobuvirus is shown in Fig. 3B 25 . Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed.", "Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed. A single RVC infection was found in seven samples and in four of these tested positive for kobuvirus at high loads 4.31 to 5.59 log 10 copies/g . A dual RVA/RVC infection was seen in two samples, but neither contained quantifiable kobuvirus loads. Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% .", "Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% . Furthermore, there was a high level of genetic variability between the 2014 Belgian porcine kobuvirus isolates, with nucleotide sequence identities ranging between 90.1 and 97.2%. A phylogenetic analysis, using the 3D gene of 17V079 and twelve Belgian isolates from 2014 Fig. 3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools.", "3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools. Veterinarians are restricted to a short list of known viruses, bacteria, parasites and management factors to define a differential diagnosis. Only the most likely cause s of the disease will be diagnostically investigated, often leading to negative, inconclusive or incomplete results. However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample.", "However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample. However, most NGS platforms require large investments and processing of the reads can only start at the end of the sequencing run. Viral metagenomics also requires extensive laboratory preparations, including centrifugation, filtration and nuclease treatment to discard bacterial and host nucleic acids that make up to the bulk of all nucleic acids present 13 . Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss.", "Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss. All these factors lead to a long turnover time between sample collection and diagnostic reporting. The third-generation sequencing device MinION ONT , holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all DNA/RNA in a sample, theoretically without needing pre-amplification of viral nucleic acids. It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets.", "It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets. Sequencing of this pooled sample with the MinION resulted in rapid identification of both viruses. Real-time analysis of the sequencing reads was not conducted, but is achievable as previously demonstrated by Greninger and colleagues using the SURPI analysis pipeline for rapid identification of human viruses from different clinical matrices 19 . Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X .", "Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X . Overall, higher sequencing depths were generated for PEDV that could indicate that sequencing of longer viral genomes is favored over smaller gene segments, as PEDV has a genome size of approximately 28 kb, and RVA gene segments are shorter 0.6 to 3.3 kb . This bias might have been introduced during the ligation of the sequencing adapters to the viral nucleic acids. It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available.", "It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available. While the technology can be useful for giving fast readouts of viruses <3 hours present in a sample, thorough validation, using well-defined virus stocks, spiking experiments in matrices e.g. feces and real clinical samples is necessary to make sure that all members of the porcine viral enteric disease complex are accurately being diagnosed. Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet.", "Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet. Real-time PCR analyses were conducted for RVA, RVC, PEDV and TGEV. Enterococcus hirae was isolated at a private diagnostic laboratory, but this bacterial species is not considered a typical cause of diarrheic disease in pigs 26 . Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted.", "Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted. However, BLASTn search options also reached high sensitivity, but at much lower time cost: 26 seconds instead of almost 24 hours. For rapid read analysis and searching for closely related non-divergent viral sequences, BLASTn or another fast methodology should thus be preferentially used. However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079.", "However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079. Astro-and enteroviruses have been detected earlier in both diarrheic and non-diarrheic feces of Belgian pigs and in feces from pigs around the globe 9,10,27 . In a recent study from Thailand, the difference in prevalence of astrovirus in diarrheic 8.4% versus non-diarrheic 4.6% piglets less than 4-weeks-old was not statistically significant. Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 .", "Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 . A recent study in 5 European countries Hungary, Spain, Germany, Austria and Sweden have indicated the widespread of porcine astroviruses in the swine population. A one hundred procent prevalence of astrovirus was found in diarrheic and non-diarrheic pigs from Austria and Spain. Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections.", "Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections. Enteroviruses have been more generally linked to neurological disorders in pigs, although they are commonly found in feces as well 11, 12, . In a study from Vietnam, no significant correlation was found between diarrhea status and presence of enterovirus G in feces 35 . The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads.", "The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads. However, reporting of a porcine kobuvirus in Belgian piglets with MinION is unique. In Belgium, kobuviruses had previously only been found in diarrheic samples of calves and young cattle in Belgium 36 . In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed.", "In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed. In one pig, an association was made between diarrheic episodes and the peak of rotavirus C shedding, a well-known enteric pathogen 37, 38 . Very interestingly, kobuvirus fecal loads were typically lower than those reported of well-described enteric viruses of which the pathogenicity has been proven using piglet infection models, such as PEDV and rotavirus . Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 .", "Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 . The finding of low kobuvirus loads in feces casts doubt over the true enteric pathogenic tropism of the virus. Hypothetically, its replication is likely not distributed across the whole villus but limited to either enterocytes at the villus' tips or to immune cells present in the gut. The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission.", "The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission. Highest rates of infection were observed in suckling piglets, compared to older pigs, in other countries . In our study, relatively long shedding of porcine kobuvirus was observed in three out of five animals, which may indicate that this virus may induce persistent infections. A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus.", "A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus. Such subclinical, yet immunosuppressive, properties have been attributed to the economically important swine pathogen porcine circovirus 47 . Of interest, one of the piglets died at the peak of kobuvirus shedding, although it was not clear if there was any causality between virus replication and the piglet's death. In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells.", "In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells. There was no evidence of cytopathogenic effect after several days of incubation. Antibodies to visualize antigen expression were not available and therefore the possibility of replication without SCIenTIFIC REPORTS | . 8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available.", "8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available. To assess more broadly the prevalence of kobuvirus in the Belgian swine industry, a retrospective analysis of diarrheic samples from suckling piglets less than two weeks old was conducted. A high proportion 40.9% of the samples n = 44 contained quantifiable viral loads ranging between 4.31 to 6.83 log 10 copies/g feces. Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made.", "Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made. However, the prevalence of kobuvirus has been widely described in pigs from several European countries The Netherlands 16.7% , Slovakia 63.4% , Hungary 81.0% , Czech Republic 87.3% , Austria 46.2% , Italy 52.4% , Germany 54.5% and Sweden 45.0% , American countries The United States 21.9% and Brazil 53.0% , African countries Kenya 14.9% and Uganda 15.5% and Asian countries Thailand 99% , South Korea 52.1% and Vietnam 29.3% . In a small proportion of these studies, statistically significant associations between prevalence of kobuvirus and diarrhea in pigs were demonstrated, such as in Hungary 54.5% prevalence in healthy pigs vs 92.3% prevalence in diarrheic pigs , Spain 47.5% healthy vs 74.4% diarrheic , Brazil 41% vs 78.4% , Thailand 19.3% vs 84.5% and Vietnam 27.6% to 40.9% 35, 45, 46, 52 . Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%.", "Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%. Furthermore, they clustered diffusely between strains from different countries around the world, indicating that strains are not distinguishable based on their geographical origin. Because most 99% of the reads generated during sequencing of the fecal sample 17V079 matched bacteriophages upon analysis with BLAST, bacteriophages may have played an important role in the pathogenesis of the diarrheic disease. De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 .", "De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 . Several contigs showed high similarities to the Bacteroides phage B124-14, found in municipal wastewater and human fecal samples. It was shown to be absent in 30 samples collected from different animal species, including pigs, and is therefore considered a human-specific phage 55, 56 . The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies.", "The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies. Interestingly, several of the contigs found also showed similarities to Escherichia phages. Two of the contigs were similar to Escherichia phages PhAPEC5 and PhAPEC7, isolated from Belgian rivers in the neighborhood of poultry houses and known to cause lytic infections in avian pathogenic Escherichia coli. Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 .", "Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 . Finally, one contig showed limited similarity to an Enterococcus phage, isolated from hospital sewage in China, while a last contig showed moderate similaraties to the bacterial Enterococcus hirae genome. This region may be a prophage, inserted in the bacterial genome. The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins.", "The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins. The new neonatal diarrhea syndrome described above shows high similarities to the disease described in the case 17V079 and it may be that bacteriophages are involved in the pathogenesis of this syndrome. So far, the role of phages has not been considered in the pathogenesis of several enteric disorders, but given the high abundance here, it should be in future studies. It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant.", "It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant. Complete overviews of all viruses and other pathogens in a sample will be given in a single readout instead of requiring different diagnostic assays. However, care should be given to the interpretation of such results, as they should only be analyzed by trained veterinarians. Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml.", "Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml. The nucleotide sequences of the 11 gene segments of this strain were resolved earlier using Sanger sequencing GenBank accession numbers: KM82070 VP1 , KM820707 VP2 , KM827014 VP3 , KM820720 VP4 , KM820728 VP6 , KM820735 VP7 , KM820742 NSP1 , KM820672 NSP2 , KM820679 NSP3 , KM820686 NSP4 and KM820693 NSP5 59 . A porcine epidemic diarrhea virus strain PEDV, CV777 isolated in Belgium in the 1970s was adapted for growth in Vero cells in the 1980s 60 . In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets.", "In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets. A diarrheic fecal sample was collected from a Belgian pig on a farm housing a total of 620 sows and using a 2-week batch-production system, with a weaning age of 23 days. Topigs Norsvin sows were crossed with Piétrain boars, producing 32. toxins Suiseng, Hipra . Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed.", "Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed. Since the spring of 2017, enteric disease started causing more severe problems accompanied with mortality on this farm, mainly in 7-days-old suckling piglets. A diarrheic fecal sample of such a piglet was investigated at a private diagnostic laboratory Dialab, Belsele, Belgium and labeled 17V079. No virological cause was found to explain the diarrheic problems on the farm. The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine .", "The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine . No other pathogens were found in this sample. As the clinical picture hinted at a viral cause for the disease, the sample was sent to the Laboratory of Virology at the Faculty of Veterinary Medicine Ghent University for further analysis. The sample tested negative for RVA, RVC, PEDV and TGEV using in-house RT-qPCR assays 25, 61, 62 . Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids.", "Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids. First, viral enrichment was done based on the NetoVIR protocol to obtain pure viral nucleic acids for sequencing library preparation 13 . MinION analyses of cell culture grown viruses RVA and PEDV were conducted at the Laboratory of Clinical Virology Rega Institute, KU Leuven , whereas the diarrheic fecal sample was analyzed at the Laboratory of Virology Faculty of Veterinary Medicine, Ghent University . RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses.", "RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses. This mixture was filtered using a 0.8 µm polyethersulphone filter for 1 min at 17,000 × g, followed by a nuclease treatment for 2 hours at 37 °C to digest free nucleic acids in the suspension: 250 µl of the sample was added to 14 µl of home-made buffer 1 M Tris, 100 mM CaCl 2 and 30 mM MgCl 2 , pH 8 , 4 µl of Benzonase Nuclease Millipore and 2 µl Micrococcal Nuclease NEB as described earlier 13 . Fourteen microliters of EDTA were added to stop the reaction, followed by extraction of nucleic acids from the viral particles using the QIAamp Viral RNA Mini Kit Qiagen . The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications.", "The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications. A 10% w/v suspension of the diarrhea was made in Minimum Essential Medium and centrifuged. The supernatant was filtered through a 0.45 µm syringe filter Sarstedt and treated with Benzonase Nuclease for 1 hour to speed up the diagnostic pipeline. Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl.", "Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl. cDNA and second strand synthesis for nanopore sequencing. Nucleic acids were heated at 95 °C for 2 min and chilled on ice to resolve secondary RNA structures and to denature double-stranded RNA. Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water.", "Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water. Primer annealing was conducted at 65 °C for 5 min, after which 4 µl Superscript IV Reaction Buffer ThermoScientific , 1 µl dithiothreitol ThermoScientific and 1 µl SuperScript IV Reverse Transcriptase ThermoScientific were added in a total reaction volume of 20 µl. The reaction conditions were as follows: 23 °C for 10 min, 50 °C for 10 min, 80 °C for 10 min and an infinite hold step at 10 °C. A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume .", "A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume . Isothermal amplification was done at 16 °C for 1 h and double-stranded nucleic acids were purified using 144 µl of magnetic AMPure XP Beads Beckman Coulter . Two washing steps with freshly prepared 70% ethanol were conducted before eluting in 52 µl nuclease-free water. Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters.", "Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters. Fifty microliters of un amplified DNA were mixed with 7 µl Ultra II End-Prep Reaction Buffer New England Biolabs and 3 µl Ultra II End-prep enzyme mix New England Biolabs , and incubated at 20 °C for 5 min and 65 °C for 5 min. Next, nucleic acids were purified using 60 µl AMPure XP Beads and eluted in 31 µl nuclease-free water. Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min.", "Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min. The sequencing library, containing double-stranded DNA with adapters ligated to the 3′ ends, was then purified using 40 µl AMPure XP beads. Two washing steps were conducted using 140 µl Adapter Bead Binding Buffer ABB, ONT before eluting in 15 µl of Elution Buffer ELB, ONT . EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT .", "EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT . Bio-informatics analyses. Raw reads were produced by MinKNOW. Live basecalling was enabled for the first experiment using MinKNOW version 1.5.5. In the second experiment, basecalling was done after the sequencing run using Albacore version 1.2.5., ONT . Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted.", "Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted. Sequences were then analyzed using different BLAST methods including BLASTn and tBLASTx BLAST version 2.6.0; e-value cut-off 1e −3 -1e −10 to compare sensitivity and run-times to detect viral sequences among the reads. A complete viral database was composed of all virus sequences in GenBank taxonomy ID 10239, containing sequences up to 17th of September 2017 . The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses.", "The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses. GraphMap version 0.5.2 and Samtools version 1.6 were used for mapping of reads against reference sequences, while Canu 1.6 was used for de novo assembly of viral genomes . VirSorter was run using the 'Viromes' database to look for phages, with the Virome Decontamination Mode on to identify phage contigs 54 . Bio-informatics analyses were executed on a local computer cluster and the high-performance computing facilities of Ghent University. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene.", "The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene. A porcine kobuvirus was discovered in the sample 17V079 using the MinION. The sequence of the 3D gene of porcine kobuvirus encodes the polymerase and is considered to be most conserved among different strains. The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 .", "The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 . The RT-PCR reaction contained 5 µl 5 × Qiagen OneStep RT-PCR Buffer, 1 µl dNTPs, 3 µl of each primer 10 µm , 7 µl nuclease-free water, 1 µl OneStep RT-PCR enzyme mix and 5 µl template RNA or water total reaction volume of 25 µl . RT-PCR conditions were as follows: 50 °C for 30 min, 95 °C for 15 min, followed by 30 cycles of amplification 94 °C for 30 s, 50 °C for 30 s and 72° for 90 s and a final extension step at 72 °C for 1 min. Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light.", "Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light. The amplicon was sent to GATC Constance, Germany for Sanger sequencing using an ABI 3730xl DNA Analyzer system. Quality control of the raw chromatograms was done using 4Peaks Nucleobytes BV, The Netherlands and BLASTn NCBI, United States . Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water.", "Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water. Three microliters of template RNA or water were added to each tube containing 17 µl mastermix. A synthetic RNA positive control 175nt was generated by RT-PCR using the primers Kobu3D_qPCR +T7_Fw and Kobu3D_qPCR_Rv, followed by in vitro transcription of this PCR product using a T7 RNA polymerase. The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s .", "The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s . Detection of SYBR Green fluorescence was done at the end of each annealing phase. A melt curve analysis was executed to assess specificity of the amplicons generated. Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene.", "Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene. Assays were valid if the efficiency over the LDR was between 90 and 110%, and R 2 of the standard curve replicates was >0.99. Quantification of the viral loads was possible if the Cq-values of two qPCR replicates fell within the LDR of the assay. Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable.", "Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable. Longitudinal investigation of kobuvirus and rotavirus shedding in suckling piglets. Upon characterization of the virome with the MinION, a longitudinal follow-up study was setup between August and September 2017. To warrant the health status of the pig stock, entrance to the farm was strictly regulated. Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer.", "Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer. Sample collection in the longitudinal field study was done in agreement with the European legislation on animal experiments. Sample collection was approved by and done in accordance to the requirements of the Local Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering of Ghent University. One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period.", "One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period. A dry cotton rectal swab Copan was collected from each individual piglet at days 1, 5, 8, 11, 14, 17, 20 and 22 after birth. The swab was placed immediately in 2 ml of viral transport medium phosphate buffered saline containing 1000 U/ml penicillin Continental Pharma, Puurs, Belgium , 1 mg/ml streptomycin Certa, Braine l′Alleud, Belgium , 1 mg/ml gentamicin Life Technologies and 0.01% v/v Fungizone Bristol-Myers Squibb, Braine l′Alleud, Belgium in a sterile 15 ml falcon tube Sarstedt and stored at −20 °C. Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs.", "Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs. Upon arrival in the Laboratory of Virology, the samples were thawed and placed on a shaker for 30 min at 4 °C to release viral particles in the transport medium. Samples were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions and purified nucleic acids were eluted in 100 µl of AVE and stored at −70 °C until RT-qPCR analysis. RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 .", "RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 . Belgian suckling pigs. Fecal samples n = 44 of diarrheic suckling piglets less than 2 weeks old were sent to a private laboratory by veterinarians Dialab, Belsele, Belgium for etiological diagnosis, as described earlier. These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 .", "These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 . RNA extraction was conducted using the QIAamp Cador Pathogen Mini Kit Qiagen as described above and RT-qPCR was done to quantify the load of kobuvirus RNA copies. Samples with a quantifiable viral load were subjected to RT-PCR to amplify the 3D polymerase gene, after which Sanger sequencing was performed. The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 .", "The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 . A maximum-likelihood phylogenetic tree was constructed with RAxML using a general time reversible model with gamma distribution 20 cats, alpha: 0.121, LogLK = 14938.461 and heuristic branch swapping 70 . Tree editing was done using Affinity Designer Serif . Pairwise distances were calculated using the p-distance model in Mega with bootstrap values set at 500 replicates." ]

[ "Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths 19.2 to 103.5X after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages.", "Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding 10 6.42 –10 7.01 copies/swab and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread n = 25, 56.8% positive of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted.", "MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded. Text: metagenomics is a valuable asset for diagnostics in pigs, leading to discovery of novel viruses and identification of porcine viral enteric disease complexes. Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective.", "Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective. Performing the necessary analyses often results in long time periods between sample arrival and diagnostic reporting, since results can only be processed after finishing the sequencing run. Third-generation sequencing using MinION Oxford Nanopore Technologies, ONT might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides.", "The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides. This signal is converted into a nucleotide sequence by computational algorithms basecalling . Since the release of MinION technology, major advances have been made in terms of the number and the quality of reads generated 15 . In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 .", "In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 . MinION has also been used as a diagnostic tool during recent Ebolavirus outbreaks in West Africa, allowing fast on-site characterization of circulating strains 17, 18 . Coupled to a laptop-based bioinformatics workflow, MinION was able to detect Chikungunya virus, Ebola virus and hepatitis C virus in less than 6 hours using earlier versions of the technology 19 . A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 .", "A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 . In veterinary virology, the use of nanopore sequencing is growing. A novel species of papillomavirus was identified in warts from giraffes, using rolling-circle amplification and nanopore sequencing 22 . The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 .", "The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 . To the author's knowledge, the present study is the first using MinION as an aid in porcine health management. This study was aimed to explore the possibilities of MinION as a rapid and easy-to-use diagnostic tool in pig health management for diagnosis of viral enteric disease complexes. The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated.", "The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated. No gene-specific or random pre-amplification of viral nucleic acids was conducted to challenge the MinION's sensitivity. A porcine kobuvirus was discovered in the latter case and a longitudinal field study was conducted hereafter to elucidate the shedding patterns of this virus. Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium.", "Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium. be performed for 243,313 reads with a mean length of 740 nucleotides. Reads with a q-score lower than 7 were filtered out, resulting in 179,015 remaining sequences mean length 816 nt for use in downstream analyses. Results of the sequencing run, including taxonomical classification and mapping of reads against PEDV and rotavirus A RVA reference genomes are shown in Fig. 1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database.", "1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database. Of these, 39.3% n = 5,985 and 10.3% n = 1,564 were assigned to viral families comprising Porcine epidemic diarrhea virus family Coronaviridae and Rotavirus family Reoviridae, subfamily Sedoreovirinae , respectively. A fraction of the reads 29.3%, n = 4,468 were assigned to order Caudovirales. These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore.", "These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore. Most reads were generated in the first twelve hours of sequencing and read accumulation was most exponential in the first three hours of sequencing Fig. 1B . PEDV and RVA sequences were extracted from the dataset and mapped against viral reference genes to calculate sequencing depths over time Fig. 1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X .", "1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X . High sequencing depths were acquired after three hours of sequencing for PEDV 103.5X and for most RVA gene segments 19.2 to 48.2X . De novo assembly was executed on the quality-filtered reads prior to identification tBLASTx to recover viral genomes. This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 .", "This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 . However, execution of de novo assembly prior to taxonomical classification tBLASTx reduced the time to identify entire viral genomes in the dataset. Virome composition of a young diarrheic piglet using nanopore sequencing. A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses.", "A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses. Different methods were used to compare the reads against a viral database using the HPC cluster of Ghent University and results are shown in Fig. 2 . Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx.", "Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx. However, there was a very high difference between wall times on the HPC cluster, with only 26 seconds of analysis time for BLASTn, versus almost 24 hours for tBLASTx. The majority of sequences were assigned to bacteriophages within the order Caudovirales and families Siphoviridae n = 3,213 to 4,163 reads , Podoviridae n = 2,506 to 3,002 reads and Myoviridae n = 912 to 1,202 reads . A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig.", "A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig. 2B . Comparison of these contigs against the GenBank database using BLAST allowed classification into four different groups. Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown .", "Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown . The longest contig with a size of 39,069 nucleotides, together with four other contigs showed similarities 95% nt identity to different Escherichia phages. As they also mapped nicely distributed across the reference genome of Escherichia phage vB_EcoP_PhAPEC7, it seems that they must also belong to one phage genome data not shown . Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome.", "Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome. Interestingly, three contigs were identified for which no similarities were found with existing viruses in GenBank, but contig 0105 mapped to the reference genome of the Enterococcus phage vB_EfaS_IME196 data not shown . These might be novel phages or divergent variants from existing phages present in GenBank. Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae.", "Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae. Kobuvirus reads were mapped against a European reference strain S-1/HUN/2007/Hungary, as shown in Fig. 2C . However, full-genome coverage at high sequencing depth was not obtained. Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated.", "Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated. The fecal shedding patterns of the different viruses and presence of diarrheic signs are shown in Fig. 3A . All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification .", "All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification . Peak shedding titers of the porcine kobuvirus varied between 6.42 and 7.01 log 10 copies/swab, which is generally lower than peak shedding observed for typical enteric viruses such as rotavirus and PEDV. Moreover, the peak of shedding was not related to diarrheic episodes, questioning the role of this virus in the pathogenesis of diarrhea on the farm. Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed.", "Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed. In contrast, there was no direct association between peak shedding of kobuvirus and diarrheic episodes. Interestingly, a peak in kobuvirus shedding was observed in piglet C at day 11 post-farrowing. This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine.", "This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine. Retrospective analysis of porcine kobuviruses shedding in Belgian diarrheic suckling pigs and phylogenetic analysis. A total of 44 diarrheic fecal samples collected in 2014 were screened for the presence of kobuvirus using the new RT-qPCR. Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification.", "Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification. The presence of RVA and RVC had been quantitatively assessed in these samples in a previous study and the occurrence of co-infections between rotaviruses and kobuvirus is shown in Fig. 3B 25 . Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed.", "Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed. A single RVC infection was found in seven samples and in four of these tested positive for kobuvirus at high loads 4.31 to 5.59 log 10 copies/g . A dual RVA/RVC infection was seen in two samples, but neither contained quantifiable kobuvirus loads. Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% .", "Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% . Furthermore, there was a high level of genetic variability between the 2014 Belgian porcine kobuvirus isolates, with nucleotide sequence identities ranging between 90.1 and 97.2%. A phylogenetic analysis, using the 3D gene of 17V079 and twelve Belgian isolates from 2014 Fig. 3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools.", "3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools. Veterinarians are restricted to a short list of known viruses, bacteria, parasites and management factors to define a differential diagnosis. Only the most likely cause s of the disease will be diagnostically investigated, often leading to negative, inconclusive or incomplete results. However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample.", "However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample. However, most NGS platforms require large investments and processing of the reads can only start at the end of the sequencing run. Viral metagenomics also requires extensive laboratory preparations, including centrifugation, filtration and nuclease treatment to discard bacterial and host nucleic acids that make up to the bulk of all nucleic acids present 13 . Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss.", "Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss. All these factors lead to a long turnover time between sample collection and diagnostic reporting. The third-generation sequencing device MinION ONT , holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all DNA/RNA in a sample, theoretically without needing pre-amplification of viral nucleic acids. It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets.", "It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets. Sequencing of this pooled sample with the MinION resulted in rapid identification of both viruses. Real-time analysis of the sequencing reads was not conducted, but is achievable as previously demonstrated by Greninger and colleagues using the SURPI analysis pipeline for rapid identification of human viruses from different clinical matrices 19 . Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X .", "Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X . Overall, higher sequencing depths were generated for PEDV that could indicate that sequencing of longer viral genomes is favored over smaller gene segments, as PEDV has a genome size of approximately 28 kb, and RVA gene segments are shorter 0.6 to 3.3 kb . This bias might have been introduced during the ligation of the sequencing adapters to the viral nucleic acids. It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available.", "It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available. While the technology can be useful for giving fast readouts of viruses <3 hours present in a sample, thorough validation, using well-defined virus stocks, spiking experiments in matrices e.g. feces and real clinical samples is necessary to make sure that all members of the porcine viral enteric disease complex are accurately being diagnosed. Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet.", "Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet. Real-time PCR analyses were conducted for RVA, RVC, PEDV and TGEV. Enterococcus hirae was isolated at a private diagnostic laboratory, but this bacterial species is not considered a typical cause of diarrheic disease in pigs 26 . Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted.", "Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted. However, BLASTn search options also reached high sensitivity, but at much lower time cost: 26 seconds instead of almost 24 hours. For rapid read analysis and searching for closely related non-divergent viral sequences, BLASTn or another fast methodology should thus be preferentially used. However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079.", "However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079. Astro-and enteroviruses have been detected earlier in both diarrheic and non-diarrheic feces of Belgian pigs and in feces from pigs around the globe 9,10,27 . In a recent study from Thailand, the difference in prevalence of astrovirus in diarrheic 8.4% versus non-diarrheic 4.6% piglets less than 4-weeks-old was not statistically significant. Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 .", "Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 . A recent study in 5 European countries Hungary, Spain, Germany, Austria and Sweden have indicated the widespread of porcine astroviruses in the swine population. A one hundred procent prevalence of astrovirus was found in diarrheic and non-diarrheic pigs from Austria and Spain. Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections.", "Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections. Enteroviruses have been more generally linked to neurological disorders in pigs, although they are commonly found in feces as well 11, 12, . In a study from Vietnam, no significant correlation was found between diarrhea status and presence of enterovirus G in feces 35 . The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads.", "The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads. However, reporting of a porcine kobuvirus in Belgian piglets with MinION is unique. In Belgium, kobuviruses had previously only been found in diarrheic samples of calves and young cattle in Belgium 36 . In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed.", "In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed. In one pig, an association was made between diarrheic episodes and the peak of rotavirus C shedding, a well-known enteric pathogen 37, 38 . Very interestingly, kobuvirus fecal loads were typically lower than those reported of well-described enteric viruses of which the pathogenicity has been proven using piglet infection models, such as PEDV and rotavirus . Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 .", "Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 . The finding of low kobuvirus loads in feces casts doubt over the true enteric pathogenic tropism of the virus. Hypothetically, its replication is likely not distributed across the whole villus but limited to either enterocytes at the villus' tips or to immune cells present in the gut. The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission.", "The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission. Highest rates of infection were observed in suckling piglets, compared to older pigs, in other countries . In our study, relatively long shedding of porcine kobuvirus was observed in three out of five animals, which may indicate that this virus may induce persistent infections. A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus.", "A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus. Such subclinical, yet immunosuppressive, properties have been attributed to the economically important swine pathogen porcine circovirus 47 . Of interest, one of the piglets died at the peak of kobuvirus shedding, although it was not clear if there was any causality between virus replication and the piglet's death. In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells.", "In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells. There was no evidence of cytopathogenic effect after several days of incubation. Antibodies to visualize antigen expression were not available and therefore the possibility of replication without SCIenTIFIC REPORTS | . 8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available.", "8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available. To assess more broadly the prevalence of kobuvirus in the Belgian swine industry, a retrospective analysis of diarrheic samples from suckling piglets less than two weeks old was conducted. A high proportion 40.9% of the samples n = 44 contained quantifiable viral loads ranging between 4.31 to 6.83 log 10 copies/g feces. Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made.", "Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made. However, the prevalence of kobuvirus has been widely described in pigs from several European countries The Netherlands 16.7% , Slovakia 63.4% , Hungary 81.0% , Czech Republic 87.3% , Austria 46.2% , Italy 52.4% , Germany 54.5% and Sweden 45.0% , American countries The United States 21.9% and Brazil 53.0% , African countries Kenya 14.9% and Uganda 15.5% and Asian countries Thailand 99% , South Korea 52.1% and Vietnam 29.3% . In a small proportion of these studies, statistically significant associations between prevalence of kobuvirus and diarrhea in pigs were demonstrated, such as in Hungary 54.5% prevalence in healthy pigs vs 92.3% prevalence in diarrheic pigs , Spain 47.5% healthy vs 74.4% diarrheic , Brazil 41% vs 78.4% , Thailand 19.3% vs 84.5% and Vietnam 27.6% to 40.9% 35, 45, 46, 52 . Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%.", "Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%. Furthermore, they clustered diffusely between strains from different countries around the world, indicating that strains are not distinguishable based on their geographical origin. Because most 99% of the reads generated during sequencing of the fecal sample 17V079 matched bacteriophages upon analysis with BLAST, bacteriophages may have played an important role in the pathogenesis of the diarrheic disease. De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 .", "De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 . Several contigs showed high similarities to the Bacteroides phage B124-14, found in municipal wastewater and human fecal samples. It was shown to be absent in 30 samples collected from different animal species, including pigs, and is therefore considered a human-specific phage 55, 56 . The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies.", "The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies. Interestingly, several of the contigs found also showed similarities to Escherichia phages. Two of the contigs were similar to Escherichia phages PhAPEC5 and PhAPEC7, isolated from Belgian rivers in the neighborhood of poultry houses and known to cause lytic infections in avian pathogenic Escherichia coli. Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 .", "Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 . Finally, one contig showed limited similarity to an Enterococcus phage, isolated from hospital sewage in China, while a last contig showed moderate similaraties to the bacterial Enterococcus hirae genome. This region may be a prophage, inserted in the bacterial genome. The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins.", "The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins. The new neonatal diarrhea syndrome described above shows high similarities to the disease described in the case 17V079 and it may be that bacteriophages are involved in the pathogenesis of this syndrome. So far, the role of phages has not been considered in the pathogenesis of several enteric disorders, but given the high abundance here, it should be in future studies. It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant.", "It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant. Complete overviews of all viruses and other pathogens in a sample will be given in a single readout instead of requiring different diagnostic assays. However, care should be given to the interpretation of such results, as they should only be analyzed by trained veterinarians. Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml.", "Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml. The nucleotide sequences of the 11 gene segments of this strain were resolved earlier using Sanger sequencing GenBank accession numbers: KM82070 VP1 , KM820707 VP2 , KM827014 VP3 , KM820720 VP4 , KM820728 VP6 , KM820735 VP7 , KM820742 NSP1 , KM820672 NSP2 , KM820679 NSP3 , KM820686 NSP4 and KM820693 NSP5 59 . A porcine epidemic diarrhea virus strain PEDV, CV777 isolated in Belgium in the 1970s was adapted for growth in Vero cells in the 1980s 60 . In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets.", "In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets. A diarrheic fecal sample was collected from a Belgian pig on a farm housing a total of 620 sows and using a 2-week batch-production system, with a weaning age of 23 days. Topigs Norsvin sows were crossed with Piétrain boars, producing 32. toxins Suiseng, Hipra . Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed.", "Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed. Since the spring of 2017, enteric disease started causing more severe problems accompanied with mortality on this farm, mainly in 7-days-old suckling piglets. A diarrheic fecal sample of such a piglet was investigated at a private diagnostic laboratory Dialab, Belsele, Belgium and labeled 17V079. No virological cause was found to explain the diarrheic problems on the farm. The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine .", "The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine . No other pathogens were found in this sample. As the clinical picture hinted at a viral cause for the disease, the sample was sent to the Laboratory of Virology at the Faculty of Veterinary Medicine Ghent University for further analysis. The sample tested negative for RVA, RVC, PEDV and TGEV using in-house RT-qPCR assays 25, 61, 62 . Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids.", "Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids. First, viral enrichment was done based on the NetoVIR protocol to obtain pure viral nucleic acids for sequencing library preparation 13 . MinION analyses of cell culture grown viruses RVA and PEDV were conducted at the Laboratory of Clinical Virology Rega Institute, KU Leuven , whereas the diarrheic fecal sample was analyzed at the Laboratory of Virology Faculty of Veterinary Medicine, Ghent University . RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses.", "RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses. This mixture was filtered using a 0.8 µm polyethersulphone filter for 1 min at 17,000 × g, followed by a nuclease treatment for 2 hours at 37 °C to digest free nucleic acids in the suspension: 250 µl of the sample was added to 14 µl of home-made buffer 1 M Tris, 100 mM CaCl 2 and 30 mM MgCl 2 , pH 8 , 4 µl of Benzonase Nuclease Millipore and 2 µl Micrococcal Nuclease NEB as described earlier 13 . Fourteen microliters of EDTA were added to stop the reaction, followed by extraction of nucleic acids from the viral particles using the QIAamp Viral RNA Mini Kit Qiagen . The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications.", "The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications. A 10% w/v suspension of the diarrhea was made in Minimum Essential Medium and centrifuged. The supernatant was filtered through a 0.45 µm syringe filter Sarstedt and treated with Benzonase Nuclease for 1 hour to speed up the diagnostic pipeline. Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl.", "Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl. cDNA and second strand synthesis for nanopore sequencing. Nucleic acids were heated at 95 °C for 2 min and chilled on ice to resolve secondary RNA structures and to denature double-stranded RNA. Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water.", "Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water. Primer annealing was conducted at 65 °C for 5 min, after which 4 µl Superscript IV Reaction Buffer ThermoScientific , 1 µl dithiothreitol ThermoScientific and 1 µl SuperScript IV Reverse Transcriptase ThermoScientific were added in a total reaction volume of 20 µl. The reaction conditions were as follows: 23 °C for 10 min, 50 °C for 10 min, 80 °C for 10 min and an infinite hold step at 10 °C. A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume .", "A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume . Isothermal amplification was done at 16 °C for 1 h and double-stranded nucleic acids were purified using 144 µl of magnetic AMPure XP Beads Beckman Coulter . Two washing steps with freshly prepared 70% ethanol were conducted before eluting in 52 µl nuclease-free water. Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters.", "Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters. Fifty microliters of un amplified DNA were mixed with 7 µl Ultra II End-Prep Reaction Buffer New England Biolabs and 3 µl Ultra II End-prep enzyme mix New England Biolabs , and incubated at 20 °C for 5 min and 65 °C for 5 min. Next, nucleic acids were purified using 60 µl AMPure XP Beads and eluted in 31 µl nuclease-free water. Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min.", "Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min. The sequencing library, containing double-stranded DNA with adapters ligated to the 3′ ends, was then purified using 40 µl AMPure XP beads. Two washing steps were conducted using 140 µl Adapter Bead Binding Buffer ABB, ONT before eluting in 15 µl of Elution Buffer ELB, ONT . EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT .", "EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT . Bio-informatics analyses. Raw reads were produced by MinKNOW. Live basecalling was enabled for the first experiment using MinKNOW version 1.5.5. In the second experiment, basecalling was done after the sequencing run using Albacore version 1.2.5., ONT . Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted.", "Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted. Sequences were then analyzed using different BLAST methods including BLASTn and tBLASTx BLAST version 2.6.0; e-value cut-off 1e −3 -1e −10 to compare sensitivity and run-times to detect viral sequences among the reads. A complete viral database was composed of all virus sequences in GenBank taxonomy ID 10239, containing sequences up to 17th of September 2017 . The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses.", "The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses. GraphMap version 0.5.2 and Samtools version 1.6 were used for mapping of reads against reference sequences, while Canu 1.6 was used for de novo assembly of viral genomes . VirSorter was run using the 'Viromes' database to look for phages, with the Virome Decontamination Mode on to identify phage contigs 54 . Bio-informatics analyses were executed on a local computer cluster and the high-performance computing facilities of Ghent University. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene.", "The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene. A porcine kobuvirus was discovered in the sample 17V079 using the MinION. The sequence of the 3D gene of porcine kobuvirus encodes the polymerase and is considered to be most conserved among different strains. The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 .", "The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 . The RT-PCR reaction contained 5 µl 5 × Qiagen OneStep RT-PCR Buffer, 1 µl dNTPs, 3 µl of each primer 10 µm , 7 µl nuclease-free water, 1 µl OneStep RT-PCR enzyme mix and 5 µl template RNA or water total reaction volume of 25 µl . RT-PCR conditions were as follows: 50 °C for 30 min, 95 °C for 15 min, followed by 30 cycles of amplification 94 °C for 30 s, 50 °C for 30 s and 72° for 90 s and a final extension step at 72 °C for 1 min. Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light.", "Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light. The amplicon was sent to GATC Constance, Germany for Sanger sequencing using an ABI 3730xl DNA Analyzer system. Quality control of the raw chromatograms was done using 4Peaks Nucleobytes BV, The Netherlands and BLASTn NCBI, United States . Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water.", "Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water. Three microliters of template RNA or water were added to each tube containing 17 µl mastermix. A synthetic RNA positive control 175nt was generated by RT-PCR using the primers Kobu3D_qPCR +T7_Fw and Kobu3D_qPCR_Rv, followed by in vitro transcription of this PCR product using a T7 RNA polymerase. The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s .", "The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s . Detection of SYBR Green fluorescence was done at the end of each annealing phase. A melt curve analysis was executed to assess specificity of the amplicons generated. Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene.", "Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene. Assays were valid if the efficiency over the LDR was between 90 and 110%, and R 2 of the standard curve replicates was >0.99. Quantification of the viral loads was possible if the Cq-values of two qPCR replicates fell within the LDR of the assay. Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable.", "Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable. Longitudinal investigation of kobuvirus and rotavirus shedding in suckling piglets. Upon characterization of the virome with the MinION, a longitudinal follow-up study was setup between August and September 2017. To warrant the health status of the pig stock, entrance to the farm was strictly regulated. Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer.", "Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer. Sample collection in the longitudinal field study was done in agreement with the European legislation on animal experiments. Sample collection was approved by and done in accordance to the requirements of the Local Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering of Ghent University. One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period.", "One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period. A dry cotton rectal swab Copan was collected from each individual piglet at days 1, 5, 8, 11, 14, 17, 20 and 22 after birth. The swab was placed immediately in 2 ml of viral transport medium phosphate buffered saline containing 1000 U/ml penicillin Continental Pharma, Puurs, Belgium , 1 mg/ml streptomycin Certa, Braine l′Alleud, Belgium , 1 mg/ml gentamicin Life Technologies and 0.01% v/v Fungizone Bristol-Myers Squibb, Braine l′Alleud, Belgium in a sterile 15 ml falcon tube Sarstedt and stored at −20 °C. Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs.", "Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs. Upon arrival in the Laboratory of Virology, the samples were thawed and placed on a shaker for 30 min at 4 °C to release viral particles in the transport medium. Samples were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions and purified nucleic acids were eluted in 100 µl of AVE and stored at −70 °C until RT-qPCR analysis. RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 .", "RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 . Belgian suckling pigs. Fecal samples n = 44 of diarrheic suckling piglets less than 2 weeks old were sent to a private laboratory by veterinarians Dialab, Belsele, Belgium for etiological diagnosis, as described earlier. These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 .", "These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 . RNA extraction was conducted using the QIAamp Cador Pathogen Mini Kit Qiagen as described above and RT-qPCR was done to quantify the load of kobuvirus RNA copies. Samples with a quantifiable viral load were subjected to RT-PCR to amplify the 3D polymerase gene, after which Sanger sequencing was performed. The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 .", "The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 . A maximum-likelihood phylogenetic tree was constructed with RAxML using a general time reversible model with gamma distribution 20 cats, alpha: 0.121, LogLK = 14938.461 and heuristic branch swapping 70 . Tree editing was done using Affinity Designer Serif . Pairwise distances were calculated using the p-distance model in Mega with bootstrap values set at 500 replicates." ]

[ "Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths 19.2 to 103.5X after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages.", "Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding 10 6.42 –10 7.01 copies/swab and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread n = 25, 56.8% positive of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted.", "MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded. Text: metagenomics is a valuable asset for diagnostics in pigs, leading to discovery of novel viruses and identification of porcine viral enteric disease complexes. Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective.", "Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective. Performing the necessary analyses often results in long time periods between sample arrival and diagnostic reporting, since results can only be processed after finishing the sequencing run. Third-generation sequencing using MinION Oxford Nanopore Technologies, ONT might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides.", "The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides. This signal is converted into a nucleotide sequence by computational algorithms basecalling . Since the release of MinION technology, major advances have been made in terms of the number and the quality of reads generated 15 . In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 .", "In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 . MinION has also been used as a diagnostic tool during recent Ebolavirus outbreaks in West Africa, allowing fast on-site characterization of circulating strains 17, 18 . Coupled to a laptop-based bioinformatics workflow, MinION was able to detect Chikungunya virus, Ebola virus and hepatitis C virus in less than 6 hours using earlier versions of the technology 19 . A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 .", "A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 . In veterinary virology, the use of nanopore sequencing is growing. A novel species of papillomavirus was identified in warts from giraffes, using rolling-circle amplification and nanopore sequencing 22 . The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 .", "The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 . To the author's knowledge, the present study is the first using MinION as an aid in porcine health management. This study was aimed to explore the possibilities of MinION as a rapid and easy-to-use diagnostic tool in pig health management for diagnosis of viral enteric disease complexes. The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated.", "The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated. No gene-specific or random pre-amplification of viral nucleic acids was conducted to challenge the MinION's sensitivity. A porcine kobuvirus was discovered in the latter case and a longitudinal field study was conducted hereafter to elucidate the shedding patterns of this virus. Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium.", "Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium. be performed for 243,313 reads with a mean length of 740 nucleotides. Reads with a q-score lower than 7 were filtered out, resulting in 179,015 remaining sequences mean length 816 nt for use in downstream analyses. Results of the sequencing run, including taxonomical classification and mapping of reads against PEDV and rotavirus A RVA reference genomes are shown in Fig. 1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database.", "1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database. Of these, 39.3% n = 5,985 and 10.3% n = 1,564 were assigned to viral families comprising Porcine epidemic diarrhea virus family Coronaviridae and Rotavirus family Reoviridae, subfamily Sedoreovirinae , respectively. A fraction of the reads 29.3%, n = 4,468 were assigned to order Caudovirales. These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore.", "These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore. Most reads were generated in the first twelve hours of sequencing and read accumulation was most exponential in the first three hours of sequencing Fig. 1B . PEDV and RVA sequences were extracted from the dataset and mapped against viral reference genes to calculate sequencing depths over time Fig. 1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X .", "1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X . High sequencing depths were acquired after three hours of sequencing for PEDV 103.5X and for most RVA gene segments 19.2 to 48.2X . De novo assembly was executed on the quality-filtered reads prior to identification tBLASTx to recover viral genomes. This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 .", "This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 . However, execution of de novo assembly prior to taxonomical classification tBLASTx reduced the time to identify entire viral genomes in the dataset. Virome composition of a young diarrheic piglet using nanopore sequencing. A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses.", "A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses. Different methods were used to compare the reads against a viral database using the HPC cluster of Ghent University and results are shown in Fig. 2 . Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx.", "Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx. However, there was a very high difference between wall times on the HPC cluster, with only 26 seconds of analysis time for BLASTn, versus almost 24 hours for tBLASTx. The majority of sequences were assigned to bacteriophages within the order Caudovirales and families Siphoviridae n = 3,213 to 4,163 reads , Podoviridae n = 2,506 to 3,002 reads and Myoviridae n = 912 to 1,202 reads . A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig.", "A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig. 2B . Comparison of these contigs against the GenBank database using BLAST allowed classification into four different groups. Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown .", "Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown . The longest contig with a size of 39,069 nucleotides, together with four other contigs showed similarities 95% nt identity to different Escherichia phages. As they also mapped nicely distributed across the reference genome of Escherichia phage vB_EcoP_PhAPEC7, it seems that they must also belong to one phage genome data not shown . Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome.", "Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome. Interestingly, three contigs were identified for which no similarities were found with existing viruses in GenBank, but contig 0105 mapped to the reference genome of the Enterococcus phage vB_EfaS_IME196 data not shown . These might be novel phages or divergent variants from existing phages present in GenBank. Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae.", "Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae. Kobuvirus reads were mapped against a European reference strain S-1/HUN/2007/Hungary, as shown in Fig. 2C . However, full-genome coverage at high sequencing depth was not obtained. Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated.", "Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated. The fecal shedding patterns of the different viruses and presence of diarrheic signs are shown in Fig. 3A . All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification .", "All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification . Peak shedding titers of the porcine kobuvirus varied between 6.42 and 7.01 log 10 copies/swab, which is generally lower than peak shedding observed for typical enteric viruses such as rotavirus and PEDV. Moreover, the peak of shedding was not related to diarrheic episodes, questioning the role of this virus in the pathogenesis of diarrhea on the farm. Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed.", "Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed. In contrast, there was no direct association between peak shedding of kobuvirus and diarrheic episodes. Interestingly, a peak in kobuvirus shedding was observed in piglet C at day 11 post-farrowing. This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine.", "This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine. Retrospective analysis of porcine kobuviruses shedding in Belgian diarrheic suckling pigs and phylogenetic analysis. A total of 44 diarrheic fecal samples collected in 2014 were screened for the presence of kobuvirus using the new RT-qPCR. Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification.", "Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification. The presence of RVA and RVC had been quantitatively assessed in these samples in a previous study and the occurrence of co-infections between rotaviruses and kobuvirus is shown in Fig. 3B 25 . Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed.", "Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed. A single RVC infection was found in seven samples and in four of these tested positive for kobuvirus at high loads 4.31 to 5.59 log 10 copies/g . A dual RVA/RVC infection was seen in two samples, but neither contained quantifiable kobuvirus loads. Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% .", "Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% . Furthermore, there was a high level of genetic variability between the 2014 Belgian porcine kobuvirus isolates, with nucleotide sequence identities ranging between 90.1 and 97.2%. A phylogenetic analysis, using the 3D gene of 17V079 and twelve Belgian isolates from 2014 Fig. 3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools.", "3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools. Veterinarians are restricted to a short list of known viruses, bacteria, parasites and management factors to define a differential diagnosis. Only the most likely cause s of the disease will be diagnostically investigated, often leading to negative, inconclusive or incomplete results. However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample.", "However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample. However, most NGS platforms require large investments and processing of the reads can only start at the end of the sequencing run. Viral metagenomics also requires extensive laboratory preparations, including centrifugation, filtration and nuclease treatment to discard bacterial and host nucleic acids that make up to the bulk of all nucleic acids present 13 . Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss.", "Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss. All these factors lead to a long turnover time between sample collection and diagnostic reporting. The third-generation sequencing device MinION ONT , holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all DNA/RNA in a sample, theoretically without needing pre-amplification of viral nucleic acids. It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets.", "It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets. Sequencing of this pooled sample with the MinION resulted in rapid identification of both viruses. Real-time analysis of the sequencing reads was not conducted, but is achievable as previously demonstrated by Greninger and colleagues using the SURPI analysis pipeline for rapid identification of human viruses from different clinical matrices 19 . Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X .", "Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X . Overall, higher sequencing depths were generated for PEDV that could indicate that sequencing of longer viral genomes is favored over smaller gene segments, as PEDV has a genome size of approximately 28 kb, and RVA gene segments are shorter 0.6 to 3.3 kb . This bias might have been introduced during the ligation of the sequencing adapters to the viral nucleic acids. It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available.", "It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available. While the technology can be useful for giving fast readouts of viruses <3 hours present in a sample, thorough validation, using well-defined virus stocks, spiking experiments in matrices e.g. feces and real clinical samples is necessary to make sure that all members of the porcine viral enteric disease complex are accurately being diagnosed. Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet.", "Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet. Real-time PCR analyses were conducted for RVA, RVC, PEDV and TGEV. Enterococcus hirae was isolated at a private diagnostic laboratory, but this bacterial species is not considered a typical cause of diarrheic disease in pigs 26 . Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted.", "Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted. However, BLASTn search options also reached high sensitivity, but at much lower time cost: 26 seconds instead of almost 24 hours. For rapid read analysis and searching for closely related non-divergent viral sequences, BLASTn or another fast methodology should thus be preferentially used. However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079.", "However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079. Astro-and enteroviruses have been detected earlier in both diarrheic and non-diarrheic feces of Belgian pigs and in feces from pigs around the globe 9,10,27 . In a recent study from Thailand, the difference in prevalence of astrovirus in diarrheic 8.4% versus non-diarrheic 4.6% piglets less than 4-weeks-old was not statistically significant. Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 .", "Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 . A recent study in 5 European countries Hungary, Spain, Germany, Austria and Sweden have indicated the widespread of porcine astroviruses in the swine population. A one hundred procent prevalence of astrovirus was found in diarrheic and non-diarrheic pigs from Austria and Spain. Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections.", "Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections. Enteroviruses have been more generally linked to neurological disorders in pigs, although they are commonly found in feces as well 11, 12, . In a study from Vietnam, no significant correlation was found between diarrhea status and presence of enterovirus G in feces 35 . The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads.", "The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads. However, reporting of a porcine kobuvirus in Belgian piglets with MinION is unique. In Belgium, kobuviruses had previously only been found in diarrheic samples of calves and young cattle in Belgium 36 . In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed.", "In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed. In one pig, an association was made between diarrheic episodes and the peak of rotavirus C shedding, a well-known enteric pathogen 37, 38 . Very interestingly, kobuvirus fecal loads were typically lower than those reported of well-described enteric viruses of which the pathogenicity has been proven using piglet infection models, such as PEDV and rotavirus . Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 .", "Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 . The finding of low kobuvirus loads in feces casts doubt over the true enteric pathogenic tropism of the virus. Hypothetically, its replication is likely not distributed across the whole villus but limited to either enterocytes at the villus' tips or to immune cells present in the gut. The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission.", "The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission. Highest rates of infection were observed in suckling piglets, compared to older pigs, in other countries . In our study, relatively long shedding of porcine kobuvirus was observed in three out of five animals, which may indicate that this virus may induce persistent infections. A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus.", "A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus. Such subclinical, yet immunosuppressive, properties have been attributed to the economically important swine pathogen porcine circovirus 47 . Of interest, one of the piglets died at the peak of kobuvirus shedding, although it was not clear if there was any causality between virus replication and the piglet's death. In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells.", "In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells. There was no evidence of cytopathogenic effect after several days of incubation. Antibodies to visualize antigen expression were not available and therefore the possibility of replication without SCIenTIFIC REPORTS | . 8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available.", "8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available. To assess more broadly the prevalence of kobuvirus in the Belgian swine industry, a retrospective analysis of diarrheic samples from suckling piglets less than two weeks old was conducted. A high proportion 40.9% of the samples n = 44 contained quantifiable viral loads ranging between 4.31 to 6.83 log 10 copies/g feces. Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made.", "Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made. However, the prevalence of kobuvirus has been widely described in pigs from several European countries The Netherlands 16.7% , Slovakia 63.4% , Hungary 81.0% , Czech Republic 87.3% , Austria 46.2% , Italy 52.4% , Germany 54.5% and Sweden 45.0% , American countries The United States 21.9% and Brazil 53.0% , African countries Kenya 14.9% and Uganda 15.5% and Asian countries Thailand 99% , South Korea 52.1% and Vietnam 29.3% . In a small proportion of these studies, statistically significant associations between prevalence of kobuvirus and diarrhea in pigs were demonstrated, such as in Hungary 54.5% prevalence in healthy pigs vs 92.3% prevalence in diarrheic pigs , Spain 47.5% healthy vs 74.4% diarrheic , Brazil 41% vs 78.4% , Thailand 19.3% vs 84.5% and Vietnam 27.6% to 40.9% 35, 45, 46, 52 . Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%.", "Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%. Furthermore, they clustered diffusely between strains from different countries around the world, indicating that strains are not distinguishable based on their geographical origin. Because most 99% of the reads generated during sequencing of the fecal sample 17V079 matched bacteriophages upon analysis with BLAST, bacteriophages may have played an important role in the pathogenesis of the diarrheic disease. De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 .", "De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 . Several contigs showed high similarities to the Bacteroides phage B124-14, found in municipal wastewater and human fecal samples. It was shown to be absent in 30 samples collected from different animal species, including pigs, and is therefore considered a human-specific phage 55, 56 . The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies.", "The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies. Interestingly, several of the contigs found also showed similarities to Escherichia phages. Two of the contigs were similar to Escherichia phages PhAPEC5 and PhAPEC7, isolated from Belgian rivers in the neighborhood of poultry houses and known to cause lytic infections in avian pathogenic Escherichia coli. Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 .", "Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 . Finally, one contig showed limited similarity to an Enterococcus phage, isolated from hospital sewage in China, while a last contig showed moderate similaraties to the bacterial Enterococcus hirae genome. This region may be a prophage, inserted in the bacterial genome. The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins.", "The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins. The new neonatal diarrhea syndrome described above shows high similarities to the disease described in the case 17V079 and it may be that bacteriophages are involved in the pathogenesis of this syndrome. So far, the role of phages has not been considered in the pathogenesis of several enteric disorders, but given the high abundance here, it should be in future studies. It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant.", "It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant. Complete overviews of all viruses and other pathogens in a sample will be given in a single readout instead of requiring different diagnostic assays. However, care should be given to the interpretation of such results, as they should only be analyzed by trained veterinarians. Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml.", "Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml. The nucleotide sequences of the 11 gene segments of this strain were resolved earlier using Sanger sequencing GenBank accession numbers: KM82070 VP1 , KM820707 VP2 , KM827014 VP3 , KM820720 VP4 , KM820728 VP6 , KM820735 VP7 , KM820742 NSP1 , KM820672 NSP2 , KM820679 NSP3 , KM820686 NSP4 and KM820693 NSP5 59 . A porcine epidemic diarrhea virus strain PEDV, CV777 isolated in Belgium in the 1970s was adapted for growth in Vero cells in the 1980s 60 . In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets.", "In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets. A diarrheic fecal sample was collected from a Belgian pig on a farm housing a total of 620 sows and using a 2-week batch-production system, with a weaning age of 23 days. Topigs Norsvin sows were crossed with Piétrain boars, producing 32. toxins Suiseng, Hipra . Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed.", "Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed. Since the spring of 2017, enteric disease started causing more severe problems accompanied with mortality on this farm, mainly in 7-days-old suckling piglets. A diarrheic fecal sample of such a piglet was investigated at a private diagnostic laboratory Dialab, Belsele, Belgium and labeled 17V079. No virological cause was found to explain the diarrheic problems on the farm. The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine .", "The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine . No other pathogens were found in this sample. As the clinical picture hinted at a viral cause for the disease, the sample was sent to the Laboratory of Virology at the Faculty of Veterinary Medicine Ghent University for further analysis. The sample tested negative for RVA, RVC, PEDV and TGEV using in-house RT-qPCR assays 25, 61, 62 . Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids.", "Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids. First, viral enrichment was done based on the NetoVIR protocol to obtain pure viral nucleic acids for sequencing library preparation 13 . MinION analyses of cell culture grown viruses RVA and PEDV were conducted at the Laboratory of Clinical Virology Rega Institute, KU Leuven , whereas the diarrheic fecal sample was analyzed at the Laboratory of Virology Faculty of Veterinary Medicine, Ghent University . RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses.", "RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses. This mixture was filtered using a 0.8 µm polyethersulphone filter for 1 min at 17,000 × g, followed by a nuclease treatment for 2 hours at 37 °C to digest free nucleic acids in the suspension: 250 µl of the sample was added to 14 µl of home-made buffer 1 M Tris, 100 mM CaCl 2 and 30 mM MgCl 2 , pH 8 , 4 µl of Benzonase Nuclease Millipore and 2 µl Micrococcal Nuclease NEB as described earlier 13 . Fourteen microliters of EDTA were added to stop the reaction, followed by extraction of nucleic acids from the viral particles using the QIAamp Viral RNA Mini Kit Qiagen . The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications.", "The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications. A 10% w/v suspension of the diarrhea was made in Minimum Essential Medium and centrifuged. The supernatant was filtered through a 0.45 µm syringe filter Sarstedt and treated with Benzonase Nuclease for 1 hour to speed up the diagnostic pipeline. Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl.", "Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl. cDNA and second strand synthesis for nanopore sequencing. Nucleic acids were heated at 95 °C for 2 min and chilled on ice to resolve secondary RNA structures and to denature double-stranded RNA. Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water.", "Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water. Primer annealing was conducted at 65 °C for 5 min, after which 4 µl Superscript IV Reaction Buffer ThermoScientific , 1 µl dithiothreitol ThermoScientific and 1 µl SuperScript IV Reverse Transcriptase ThermoScientific were added in a total reaction volume of 20 µl. The reaction conditions were as follows: 23 °C for 10 min, 50 °C for 10 min, 80 °C for 10 min and an infinite hold step at 10 °C. A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume .", "A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume . Isothermal amplification was done at 16 °C for 1 h and double-stranded nucleic acids were purified using 144 µl of magnetic AMPure XP Beads Beckman Coulter . Two washing steps with freshly prepared 70% ethanol were conducted before eluting in 52 µl nuclease-free water. Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters.", "Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters. Fifty microliters of un amplified DNA were mixed with 7 µl Ultra II End-Prep Reaction Buffer New England Biolabs and 3 µl Ultra II End-prep enzyme mix New England Biolabs , and incubated at 20 °C for 5 min and 65 °C for 5 min. Next, nucleic acids were purified using 60 µl AMPure XP Beads and eluted in 31 µl nuclease-free water. Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min.", "Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min. The sequencing library, containing double-stranded DNA with adapters ligated to the 3′ ends, was then purified using 40 µl AMPure XP beads. Two washing steps were conducted using 140 µl Adapter Bead Binding Buffer ABB, ONT before eluting in 15 µl of Elution Buffer ELB, ONT . EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT .", "EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT . Bio-informatics analyses. Raw reads were produced by MinKNOW. Live basecalling was enabled for the first experiment using MinKNOW version 1.5.5. In the second experiment, basecalling was done after the sequencing run using Albacore version 1.2.5., ONT . Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted.", "Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted. Sequences were then analyzed using different BLAST methods including BLASTn and tBLASTx BLAST version 2.6.0; e-value cut-off 1e −3 -1e −10 to compare sensitivity and run-times to detect viral sequences among the reads. A complete viral database was composed of all virus sequences in GenBank taxonomy ID 10239, containing sequences up to 17th of September 2017 . The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses.", "The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses. GraphMap version 0.5.2 and Samtools version 1.6 were used for mapping of reads against reference sequences, while Canu 1.6 was used for de novo assembly of viral genomes . VirSorter was run using the 'Viromes' database to look for phages, with the Virome Decontamination Mode on to identify phage contigs 54 . Bio-informatics analyses were executed on a local computer cluster and the high-performance computing facilities of Ghent University. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene.", "The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene. A porcine kobuvirus was discovered in the sample 17V079 using the MinION. The sequence of the 3D gene of porcine kobuvirus encodes the polymerase and is considered to be most conserved among different strains. The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 .", "The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 . The RT-PCR reaction contained 5 µl 5 × Qiagen OneStep RT-PCR Buffer, 1 µl dNTPs, 3 µl of each primer 10 µm , 7 µl nuclease-free water, 1 µl OneStep RT-PCR enzyme mix and 5 µl template RNA or water total reaction volume of 25 µl . RT-PCR conditions were as follows: 50 °C for 30 min, 95 °C for 15 min, followed by 30 cycles of amplification 94 °C for 30 s, 50 °C for 30 s and 72° for 90 s and a final extension step at 72 °C for 1 min. Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light.", "Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light. The amplicon was sent to GATC Constance, Germany for Sanger sequencing using an ABI 3730xl DNA Analyzer system. Quality control of the raw chromatograms was done using 4Peaks Nucleobytes BV, The Netherlands and BLASTn NCBI, United States . Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water.", "Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water. Three microliters of template RNA or water were added to each tube containing 17 µl mastermix. A synthetic RNA positive control 175nt was generated by RT-PCR using the primers Kobu3D_qPCR +T7_Fw and Kobu3D_qPCR_Rv, followed by in vitro transcription of this PCR product using a T7 RNA polymerase. The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s .", "The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s . Detection of SYBR Green fluorescence was done at the end of each annealing phase. A melt curve analysis was executed to assess specificity of the amplicons generated. Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene.", "Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene. Assays were valid if the efficiency over the LDR was between 90 and 110%, and R 2 of the standard curve replicates was >0.99. Quantification of the viral loads was possible if the Cq-values of two qPCR replicates fell within the LDR of the assay. Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable.", "Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable. Longitudinal investigation of kobuvirus and rotavirus shedding in suckling piglets. Upon characterization of the virome with the MinION, a longitudinal follow-up study was setup between August and September 2017. To warrant the health status of the pig stock, entrance to the farm was strictly regulated. Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer.", "Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer. Sample collection in the longitudinal field study was done in agreement with the European legislation on animal experiments. Sample collection was approved by and done in accordance to the requirements of the Local Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering of Ghent University. One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period.", "One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period. A dry cotton rectal swab Copan was collected from each individual piglet at days 1, 5, 8, 11, 14, 17, 20 and 22 after birth. The swab was placed immediately in 2 ml of viral transport medium phosphate buffered saline containing 1000 U/ml penicillin Continental Pharma, Puurs, Belgium , 1 mg/ml streptomycin Certa, Braine l′Alleud, Belgium , 1 mg/ml gentamicin Life Technologies and 0.01% v/v Fungizone Bristol-Myers Squibb, Braine l′Alleud, Belgium in a sterile 15 ml falcon tube Sarstedt and stored at −20 °C. Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs.", "Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs. Upon arrival in the Laboratory of Virology, the samples were thawed and placed on a shaker for 30 min at 4 °C to release viral particles in the transport medium. Samples were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions and purified nucleic acids were eluted in 100 µl of AVE and stored at −70 °C until RT-qPCR analysis. RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 .", "RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 . Belgian suckling pigs. Fecal samples n = 44 of diarrheic suckling piglets less than 2 weeks old were sent to a private laboratory by veterinarians Dialab, Belsele, Belgium for etiological diagnosis, as described earlier. These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 .", "These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 . RNA extraction was conducted using the QIAamp Cador Pathogen Mini Kit Qiagen as described above and RT-qPCR was done to quantify the load of kobuvirus RNA copies. Samples with a quantifiable viral load were subjected to RT-PCR to amplify the 3D polymerase gene, after which Sanger sequencing was performed. The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 .", "The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 . A maximum-likelihood phylogenetic tree was constructed with RAxML using a general time reversible model with gamma distribution 20 cats, alpha: 0.121, LogLK = 14938.461 and heuristic branch swapping 70 . Tree editing was done using Affinity Designer Serif . Pairwise distances were calculated using the p-distance model in Mega with bootstrap values set at 500 replicates." ]

[ "Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths 19.2 to 103.5X after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages.", "Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding 10 6.42 –10 7.01 copies/swab and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread n = 25, 56.8% positive of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted.", "MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded. Text: metagenomics is a valuable asset for diagnostics in pigs, leading to discovery of novel viruses and identification of porcine viral enteric disease complexes. Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective.", "Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective. Performing the necessary analyses often results in long time periods between sample arrival and diagnostic reporting, since results can only be processed after finishing the sequencing run. Third-generation sequencing using MinION Oxford Nanopore Technologies, ONT might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides.", "The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides. This signal is converted into a nucleotide sequence by computational algorithms basecalling . Since the release of MinION technology, major advances have been made in terms of the number and the quality of reads generated 15 . In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 .", "In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 . MinION has also been used as a diagnostic tool during recent Ebolavirus outbreaks in West Africa, allowing fast on-site characterization of circulating strains 17, 18 . Coupled to a laptop-based bioinformatics workflow, MinION was able to detect Chikungunya virus, Ebola virus and hepatitis C virus in less than 6 hours using earlier versions of the technology 19 . A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 .", "A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 . In veterinary virology, the use of nanopore sequencing is growing. A novel species of papillomavirus was identified in warts from giraffes, using rolling-circle amplification and nanopore sequencing 22 . The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 .", "The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 . To the author's knowledge, the present study is the first using MinION as an aid in porcine health management. This study was aimed to explore the possibilities of MinION as a rapid and easy-to-use diagnostic tool in pig health management for diagnosis of viral enteric disease complexes. The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated.", "The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated. No gene-specific or random pre-amplification of viral nucleic acids was conducted to challenge the MinION's sensitivity. A porcine kobuvirus was discovered in the latter case and a longitudinal field study was conducted hereafter to elucidate the shedding patterns of this virus. Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium.", "Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium. be performed for 243,313 reads with a mean length of 740 nucleotides. Reads with a q-score lower than 7 were filtered out, resulting in 179,015 remaining sequences mean length 816 nt for use in downstream analyses. Results of the sequencing run, including taxonomical classification and mapping of reads against PEDV and rotavirus A RVA reference genomes are shown in Fig. 1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database.", "1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database. Of these, 39.3% n = 5,985 and 10.3% n = 1,564 were assigned to viral families comprising Porcine epidemic diarrhea virus family Coronaviridae and Rotavirus family Reoviridae, subfamily Sedoreovirinae , respectively. A fraction of the reads 29.3%, n = 4,468 were assigned to order Caudovirales. These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore.", "These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore. Most reads were generated in the first twelve hours of sequencing and read accumulation was most exponential in the first three hours of sequencing Fig. 1B . PEDV and RVA sequences were extracted from the dataset and mapped against viral reference genes to calculate sequencing depths over time Fig. 1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X .", "1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X . High sequencing depths were acquired after three hours of sequencing for PEDV 103.5X and for most RVA gene segments 19.2 to 48.2X . De novo assembly was executed on the quality-filtered reads prior to identification tBLASTx to recover viral genomes. This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 .", "This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 . However, execution of de novo assembly prior to taxonomical classification tBLASTx reduced the time to identify entire viral genomes in the dataset. Virome composition of a young diarrheic piglet using nanopore sequencing. A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses.", "A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses. Different methods were used to compare the reads against a viral database using the HPC cluster of Ghent University and results are shown in Fig. 2 . Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx.", "Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx. However, there was a very high difference between wall times on the HPC cluster, with only 26 seconds of analysis time for BLASTn, versus almost 24 hours for tBLASTx. The majority of sequences were assigned to bacteriophages within the order Caudovirales and families Siphoviridae n = 3,213 to 4,163 reads , Podoviridae n = 2,506 to 3,002 reads and Myoviridae n = 912 to 1,202 reads . A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig.", "A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig. 2B . Comparison of these contigs against the GenBank database using BLAST allowed classification into four different groups. Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown .", "Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown . The longest contig with a size of 39,069 nucleotides, together with four other contigs showed similarities 95% nt identity to different Escherichia phages. As they also mapped nicely distributed across the reference genome of Escherichia phage vB_EcoP_PhAPEC7, it seems that they must also belong to one phage genome data not shown . Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome.", "Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome. Interestingly, three contigs were identified for which no similarities were found with existing viruses in GenBank, but contig 0105 mapped to the reference genome of the Enterococcus phage vB_EfaS_IME196 data not shown . These might be novel phages or divergent variants from existing phages present in GenBank. Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae.", "Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae. Kobuvirus reads were mapped against a European reference strain S-1/HUN/2007/Hungary, as shown in Fig. 2C . However, full-genome coverage at high sequencing depth was not obtained. Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated.", "Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated. The fecal shedding patterns of the different viruses and presence of diarrheic signs are shown in Fig. 3A . All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification .", "All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification . Peak shedding titers of the porcine kobuvirus varied between 6.42 and 7.01 log 10 copies/swab, which is generally lower than peak shedding observed for typical enteric viruses such as rotavirus and PEDV. Moreover, the peak of shedding was not related to diarrheic episodes, questioning the role of this virus in the pathogenesis of diarrhea on the farm. Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed.", "Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed. In contrast, there was no direct association between peak shedding of kobuvirus and diarrheic episodes. Interestingly, a peak in kobuvirus shedding was observed in piglet C at day 11 post-farrowing. This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine.", "This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine. Retrospective analysis of porcine kobuviruses shedding in Belgian diarrheic suckling pigs and phylogenetic analysis. A total of 44 diarrheic fecal samples collected in 2014 were screened for the presence of kobuvirus using the new RT-qPCR. Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification.", "Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification. The presence of RVA and RVC had been quantitatively assessed in these samples in a previous study and the occurrence of co-infections between rotaviruses and kobuvirus is shown in Fig. 3B 25 . Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed.", "Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed. A single RVC infection was found in seven samples and in four of these tested positive for kobuvirus at high loads 4.31 to 5.59 log 10 copies/g . A dual RVA/RVC infection was seen in two samples, but neither contained quantifiable kobuvirus loads. Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% .", "Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% . Furthermore, there was a high level of genetic variability between the 2014 Belgian porcine kobuvirus isolates, with nucleotide sequence identities ranging between 90.1 and 97.2%. A phylogenetic analysis, using the 3D gene of 17V079 and twelve Belgian isolates from 2014 Fig. 3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools.", "3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools. Veterinarians are restricted to a short list of known viruses, bacteria, parasites and management factors to define a differential diagnosis. Only the most likely cause s of the disease will be diagnostically investigated, often leading to negative, inconclusive or incomplete results. However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample.", "However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample. However, most NGS platforms require large investments and processing of the reads can only start at the end of the sequencing run. Viral metagenomics also requires extensive laboratory preparations, including centrifugation, filtration and nuclease treatment to discard bacterial and host nucleic acids that make up to the bulk of all nucleic acids present 13 . Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss.", "Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss. All these factors lead to a long turnover time between sample collection and diagnostic reporting. The third-generation sequencing device MinION ONT , holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all DNA/RNA in a sample, theoretically without needing pre-amplification of viral nucleic acids. It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets.", "It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets. Sequencing of this pooled sample with the MinION resulted in rapid identification of both viruses. Real-time analysis of the sequencing reads was not conducted, but is achievable as previously demonstrated by Greninger and colleagues using the SURPI analysis pipeline for rapid identification of human viruses from different clinical matrices 19 . Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X .", "Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X . Overall, higher sequencing depths were generated for PEDV that could indicate that sequencing of longer viral genomes is favored over smaller gene segments, as PEDV has a genome size of approximately 28 kb, and RVA gene segments are shorter 0.6 to 3.3 kb . This bias might have been introduced during the ligation of the sequencing adapters to the viral nucleic acids. It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available.", "It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available. While the technology can be useful for giving fast readouts of viruses <3 hours present in a sample, thorough validation, using well-defined virus stocks, spiking experiments in matrices e.g. feces and real clinical samples is necessary to make sure that all members of the porcine viral enteric disease complex are accurately being diagnosed. Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet.", "Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet. Real-time PCR analyses were conducted for RVA, RVC, PEDV and TGEV. Enterococcus hirae was isolated at a private diagnostic laboratory, but this bacterial species is not considered a typical cause of diarrheic disease in pigs 26 . Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted.", "Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted. However, BLASTn search options also reached high sensitivity, but at much lower time cost: 26 seconds instead of almost 24 hours. For rapid read analysis and searching for closely related non-divergent viral sequences, BLASTn or another fast methodology should thus be preferentially used. However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079.", "However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079. Astro-and enteroviruses have been detected earlier in both diarrheic and non-diarrheic feces of Belgian pigs and in feces from pigs around the globe 9,10,27 . In a recent study from Thailand, the difference in prevalence of astrovirus in diarrheic 8.4% versus non-diarrheic 4.6% piglets less than 4-weeks-old was not statistically significant. Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 .", "Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 . A recent study in 5 European countries Hungary, Spain, Germany, Austria and Sweden have indicated the widespread of porcine astroviruses in the swine population. A one hundred procent prevalence of astrovirus was found in diarrheic and non-diarrheic pigs from Austria and Spain. Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections.", "Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections. Enteroviruses have been more generally linked to neurological disorders in pigs, although they are commonly found in feces as well 11, 12, . In a study from Vietnam, no significant correlation was found between diarrhea status and presence of enterovirus G in feces 35 . The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads.", "The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads. However, reporting of a porcine kobuvirus in Belgian piglets with MinION is unique. In Belgium, kobuviruses had previously only been found in diarrheic samples of calves and young cattle in Belgium 36 . In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed.", "In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed. In one pig, an association was made between diarrheic episodes and the peak of rotavirus C shedding, a well-known enteric pathogen 37, 38 . Very interestingly, kobuvirus fecal loads were typically lower than those reported of well-described enteric viruses of which the pathogenicity has been proven using piglet infection models, such as PEDV and rotavirus . Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 .", "Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 . The finding of low kobuvirus loads in feces casts doubt over the true enteric pathogenic tropism of the virus. Hypothetically, its replication is likely not distributed across the whole villus but limited to either enterocytes at the villus' tips or to immune cells present in the gut. The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission.", "The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission. Highest rates of infection were observed in suckling piglets, compared to older pigs, in other countries . In our study, relatively long shedding of porcine kobuvirus was observed in three out of five animals, which may indicate that this virus may induce persistent infections. A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus.", "A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus. Such subclinical, yet immunosuppressive, properties have been attributed to the economically important swine pathogen porcine circovirus 47 . Of interest, one of the piglets died at the peak of kobuvirus shedding, although it was not clear if there was any causality between virus replication and the piglet's death. In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells.", "In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells. There was no evidence of cytopathogenic effect after several days of incubation. Antibodies to visualize antigen expression were not available and therefore the possibility of replication without SCIenTIFIC REPORTS | . 8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available.", "8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available. To assess more broadly the prevalence of kobuvirus in the Belgian swine industry, a retrospective analysis of diarrheic samples from suckling piglets less than two weeks old was conducted. A high proportion 40.9% of the samples n = 44 contained quantifiable viral loads ranging between 4.31 to 6.83 log 10 copies/g feces. Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made.", "Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made. However, the prevalence of kobuvirus has been widely described in pigs from several European countries The Netherlands 16.7% , Slovakia 63.4% , Hungary 81.0% , Czech Republic 87.3% , Austria 46.2% , Italy 52.4% , Germany 54.5% and Sweden 45.0% , American countries The United States 21.9% and Brazil 53.0% , African countries Kenya 14.9% and Uganda 15.5% and Asian countries Thailand 99% , South Korea 52.1% and Vietnam 29.3% . In a small proportion of these studies, statistically significant associations between prevalence of kobuvirus and diarrhea in pigs were demonstrated, such as in Hungary 54.5% prevalence in healthy pigs vs 92.3% prevalence in diarrheic pigs , Spain 47.5% healthy vs 74.4% diarrheic , Brazil 41% vs 78.4% , Thailand 19.3% vs 84.5% and Vietnam 27.6% to 40.9% 35, 45, 46, 52 . Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%.", "Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%. Furthermore, they clustered diffusely between strains from different countries around the world, indicating that strains are not distinguishable based on their geographical origin. Because most 99% of the reads generated during sequencing of the fecal sample 17V079 matched bacteriophages upon analysis with BLAST, bacteriophages may have played an important role in the pathogenesis of the diarrheic disease. De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 .", "De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 . Several contigs showed high similarities to the Bacteroides phage B124-14, found in municipal wastewater and human fecal samples. It was shown to be absent in 30 samples collected from different animal species, including pigs, and is therefore considered a human-specific phage 55, 56 . The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies.", "The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies. Interestingly, several of the contigs found also showed similarities to Escherichia phages. Two of the contigs were similar to Escherichia phages PhAPEC5 and PhAPEC7, isolated from Belgian rivers in the neighborhood of poultry houses and known to cause lytic infections in avian pathogenic Escherichia coli. Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 .", "Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 . Finally, one contig showed limited similarity to an Enterococcus phage, isolated from hospital sewage in China, while a last contig showed moderate similaraties to the bacterial Enterococcus hirae genome. This region may be a prophage, inserted in the bacterial genome. The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins.", "The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins. The new neonatal diarrhea syndrome described above shows high similarities to the disease described in the case 17V079 and it may be that bacteriophages are involved in the pathogenesis of this syndrome. So far, the role of phages has not been considered in the pathogenesis of several enteric disorders, but given the high abundance here, it should be in future studies. It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant.", "It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant. Complete overviews of all viruses and other pathogens in a sample will be given in a single readout instead of requiring different diagnostic assays. However, care should be given to the interpretation of such results, as they should only be analyzed by trained veterinarians. Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml.", "Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml. The nucleotide sequences of the 11 gene segments of this strain were resolved earlier using Sanger sequencing GenBank accession numbers: KM82070 VP1 , KM820707 VP2 , KM827014 VP3 , KM820720 VP4 , KM820728 VP6 , KM820735 VP7 , KM820742 NSP1 , KM820672 NSP2 , KM820679 NSP3 , KM820686 NSP4 and KM820693 NSP5 59 . A porcine epidemic diarrhea virus strain PEDV, CV777 isolated in Belgium in the 1970s was adapted for growth in Vero cells in the 1980s 60 . In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets.", "In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets. A diarrheic fecal sample was collected from a Belgian pig on a farm housing a total of 620 sows and using a 2-week batch-production system, with a weaning age of 23 days. Topigs Norsvin sows were crossed with Piétrain boars, producing 32. toxins Suiseng, Hipra . Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed.", "Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed. Since the spring of 2017, enteric disease started causing more severe problems accompanied with mortality on this farm, mainly in 7-days-old suckling piglets. A diarrheic fecal sample of such a piglet was investigated at a private diagnostic laboratory Dialab, Belsele, Belgium and labeled 17V079. No virological cause was found to explain the diarrheic problems on the farm. The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine .", "The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine . No other pathogens were found in this sample. As the clinical picture hinted at a viral cause for the disease, the sample was sent to the Laboratory of Virology at the Faculty of Veterinary Medicine Ghent University for further analysis. The sample tested negative for RVA, RVC, PEDV and TGEV using in-house RT-qPCR assays 25, 61, 62 . Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids.", "Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids. First, viral enrichment was done based on the NetoVIR protocol to obtain pure viral nucleic acids for sequencing library preparation 13 . MinION analyses of cell culture grown viruses RVA and PEDV were conducted at the Laboratory of Clinical Virology Rega Institute, KU Leuven , whereas the diarrheic fecal sample was analyzed at the Laboratory of Virology Faculty of Veterinary Medicine, Ghent University . RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses.", "RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses. This mixture was filtered using a 0.8 µm polyethersulphone filter for 1 min at 17,000 × g, followed by a nuclease treatment for 2 hours at 37 °C to digest free nucleic acids in the suspension: 250 µl of the sample was added to 14 µl of home-made buffer 1 M Tris, 100 mM CaCl 2 and 30 mM MgCl 2 , pH 8 , 4 µl of Benzonase Nuclease Millipore and 2 µl Micrococcal Nuclease NEB as described earlier 13 . Fourteen microliters of EDTA were added to stop the reaction, followed by extraction of nucleic acids from the viral particles using the QIAamp Viral RNA Mini Kit Qiagen . The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications.", "The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications. A 10% w/v suspension of the diarrhea was made in Minimum Essential Medium and centrifuged. The supernatant was filtered through a 0.45 µm syringe filter Sarstedt and treated with Benzonase Nuclease for 1 hour to speed up the diagnostic pipeline. Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl.", "Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl. cDNA and second strand synthesis for nanopore sequencing. Nucleic acids were heated at 95 °C for 2 min and chilled on ice to resolve secondary RNA structures and to denature double-stranded RNA. Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water.", "Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water. Primer annealing was conducted at 65 °C for 5 min, after which 4 µl Superscript IV Reaction Buffer ThermoScientific , 1 µl dithiothreitol ThermoScientific and 1 µl SuperScript IV Reverse Transcriptase ThermoScientific were added in a total reaction volume of 20 µl. The reaction conditions were as follows: 23 °C for 10 min, 50 °C for 10 min, 80 °C for 10 min and an infinite hold step at 10 °C. A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume .", "A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume . Isothermal amplification was done at 16 °C for 1 h and double-stranded nucleic acids were purified using 144 µl of magnetic AMPure XP Beads Beckman Coulter . Two washing steps with freshly prepared 70% ethanol were conducted before eluting in 52 µl nuclease-free water. Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters.", "Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters. Fifty microliters of un amplified DNA were mixed with 7 µl Ultra II End-Prep Reaction Buffer New England Biolabs and 3 µl Ultra II End-prep enzyme mix New England Biolabs , and incubated at 20 °C for 5 min and 65 °C for 5 min. Next, nucleic acids were purified using 60 µl AMPure XP Beads and eluted in 31 µl nuclease-free water. Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min.", "Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min. The sequencing library, containing double-stranded DNA with adapters ligated to the 3′ ends, was then purified using 40 µl AMPure XP beads. Two washing steps were conducted using 140 µl Adapter Bead Binding Buffer ABB, ONT before eluting in 15 µl of Elution Buffer ELB, ONT . EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT .", "EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT . Bio-informatics analyses. Raw reads were produced by MinKNOW. Live basecalling was enabled for the first experiment using MinKNOW version 1.5.5. In the second experiment, basecalling was done after the sequencing run using Albacore version 1.2.5., ONT . Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted.", "Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted. Sequences were then analyzed using different BLAST methods including BLASTn and tBLASTx BLAST version 2.6.0; e-value cut-off 1e −3 -1e −10 to compare sensitivity and run-times to detect viral sequences among the reads. A complete viral database was composed of all virus sequences in GenBank taxonomy ID 10239, containing sequences up to 17th of September 2017 . The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses.", "The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses. GraphMap version 0.5.2 and Samtools version 1.6 were used for mapping of reads against reference sequences, while Canu 1.6 was used for de novo assembly of viral genomes . VirSorter was run using the 'Viromes' database to look for phages, with the Virome Decontamination Mode on to identify phage contigs 54 . Bio-informatics analyses were executed on a local computer cluster and the high-performance computing facilities of Ghent University. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene.", "The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene. A porcine kobuvirus was discovered in the sample 17V079 using the MinION. The sequence of the 3D gene of porcine kobuvirus encodes the polymerase and is considered to be most conserved among different strains. The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 .", "The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 . The RT-PCR reaction contained 5 µl 5 × Qiagen OneStep RT-PCR Buffer, 1 µl dNTPs, 3 µl of each primer 10 µm , 7 µl nuclease-free water, 1 µl OneStep RT-PCR enzyme mix and 5 µl template RNA or water total reaction volume of 25 µl . RT-PCR conditions were as follows: 50 °C for 30 min, 95 °C for 15 min, followed by 30 cycles of amplification 94 °C for 30 s, 50 °C for 30 s and 72° for 90 s and a final extension step at 72 °C for 1 min. Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light.", "Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light. The amplicon was sent to GATC Constance, Germany for Sanger sequencing using an ABI 3730xl DNA Analyzer system. Quality control of the raw chromatograms was done using 4Peaks Nucleobytes BV, The Netherlands and BLASTn NCBI, United States . Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water.", "Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water. Three microliters of template RNA or water were added to each tube containing 17 µl mastermix. A synthetic RNA positive control 175nt was generated by RT-PCR using the primers Kobu3D_qPCR +T7_Fw and Kobu3D_qPCR_Rv, followed by in vitro transcription of this PCR product using a T7 RNA polymerase. The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s .", "The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s . Detection of SYBR Green fluorescence was done at the end of each annealing phase. A melt curve analysis was executed to assess specificity of the amplicons generated. Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene.", "Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene. Assays were valid if the efficiency over the LDR was between 90 and 110%, and R 2 of the standard curve replicates was >0.99. Quantification of the viral loads was possible if the Cq-values of two qPCR replicates fell within the LDR of the assay. Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable.", "Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable. Longitudinal investigation of kobuvirus and rotavirus shedding in suckling piglets. Upon characterization of the virome with the MinION, a longitudinal follow-up study was setup between August and September 2017. To warrant the health status of the pig stock, entrance to the farm was strictly regulated. Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer.", "Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer. Sample collection in the longitudinal field study was done in agreement with the European legislation on animal experiments. Sample collection was approved by and done in accordance to the requirements of the Local Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering of Ghent University. One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period.", "One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period. A dry cotton rectal swab Copan was collected from each individual piglet at days 1, 5, 8, 11, 14, 17, 20 and 22 after birth. The swab was placed immediately in 2 ml of viral transport medium phosphate buffered saline containing 1000 U/ml penicillin Continental Pharma, Puurs, Belgium , 1 mg/ml streptomycin Certa, Braine l′Alleud, Belgium , 1 mg/ml gentamicin Life Technologies and 0.01% v/v Fungizone Bristol-Myers Squibb, Braine l′Alleud, Belgium in a sterile 15 ml falcon tube Sarstedt and stored at −20 °C. Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs.", "Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs. Upon arrival in the Laboratory of Virology, the samples were thawed and placed on a shaker for 30 min at 4 °C to release viral particles in the transport medium. Samples were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions and purified nucleic acids were eluted in 100 µl of AVE and stored at −70 °C until RT-qPCR analysis. RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 .", "RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 . Belgian suckling pigs. Fecal samples n = 44 of diarrheic suckling piglets less than 2 weeks old were sent to a private laboratory by veterinarians Dialab, Belsele, Belgium for etiological diagnosis, as described earlier. These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 .", "These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 . RNA extraction was conducted using the QIAamp Cador Pathogen Mini Kit Qiagen as described above and RT-qPCR was done to quantify the load of kobuvirus RNA copies. Samples with a quantifiable viral load were subjected to RT-PCR to amplify the 3D polymerase gene, after which Sanger sequencing was performed. The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 .", "The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 . A maximum-likelihood phylogenetic tree was constructed with RAxML using a general time reversible model with gamma distribution 20 cats, alpha: 0.121, LogLK = 14938.461 and heuristic branch swapping 70 . Tree editing was done using Affinity Designer Serif . Pairwise distances were calculated using the p-distance model in Mega with bootstrap values set at 500 replicates." ]

[ "Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for viral diagnostics were investigated here. First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths 19.2 to 103.5X after 3 h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages.", "Almost all reads 99% belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding 10 6.42 –10 7.01 copies/swab and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread n = 25, 56.8% positive of genetically moderately related kobuviruses among Belgian diarrheic piglets. MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted.", "MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded. Text: metagenomics is a valuable asset for diagnostics in pigs, leading to discovery of novel viruses and identification of porcine viral enteric disease complexes. Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective.", "Although standardized procedures have been developed to study viral metagenomes in fecal samples, they still require an extensive sample preparation, including random or targeted pre-amplification of viral genomes present in the sample 13 . Most sequencing platforms still require capital investments and high sample turnover rates to be cost-effective. Performing the necessary analyses often results in long time periods between sample arrival and diagnostic reporting, since results can only be processed after finishing the sequencing run. Third-generation sequencing using MinION Oxford Nanopore Technologies, ONT might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides.", "The flowcells used for sequencing consist of a membrane containing multiple CsgG nanopore proteins from Escherichia coli 14 . An ion current is established through this pore resulting in typical current changes upon passage of specific nucleotides. This signal is converted into a nucleotide sequence by computational algorithms basecalling . Since the release of MinION technology, major advances have been made in terms of the number and the quality of reads generated 15 . In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 .", "In the field of virology, the technology has mainly been applied in human medicine. Using nanopore sequencing, it was possible to distinguish three poxviruses with 98% nucleotide similarity at strain level 16 . MinION has also been used as a diagnostic tool during recent Ebolavirus outbreaks in West Africa, allowing fast on-site characterization of circulating strains 17, 18 . Coupled to a laptop-based bioinformatics workflow, MinION was able to detect Chikungunya virus, Ebola virus and hepatitis C virus in less than 6 hours using earlier versions of the technology 19 . A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 .", "A multiplex PCR method for complete on-site Zikavirus genome sequencing in samples with low viral loads has recently been developed by Quick and coworkers 20 . Partial dengue virus genomes were isothermally amplified followed by sequencing, allowing classification of strains in serotypes 21 . In veterinary virology, the use of nanopore sequencing is growing. A novel species of papillomavirus was identified in warts from giraffes, using rolling-circle amplification and nanopore sequencing 22 . The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 .", "The entire genome of a parapoxvirus isolated from a seal was obtained by combining data from Illumina next-generation sequencing with nanopore sequencing data 23 . One study has reported the detection of Venezuelan equine encephalitis virus from unamplified cDNA created from poly-A tailed RNA using cell culture grown viruses 24 . To the author's knowledge, the present study is the first using MinION as an aid in porcine health management. This study was aimed to explore the possibilities of MinION as a rapid and easy-to-use diagnostic tool in pig health management for diagnosis of viral enteric disease complexes. The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated.", "The ability to detect high loads of cell culture-grown rotavirus and coronavirus, mimicking shedding quantities observed in diarrheic piglets, was evaluated. In a second case, the ability to detect novel viruses in diarrheic feces of a one-week-old piglet with diarrhea was investigated. No gene-specific or random pre-amplification of viral nucleic acids was conducted to challenge the MinION's sensitivity. A porcine kobuvirus was discovered in the latter case and a longitudinal field study was conducted hereafter to elucidate the shedding patterns of this virus. Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium.", "Moreover, archival . fecal samples from diarrheic suckling piglets less than two weeks old were investigated for the presence of kobuviruses, to study their epidemiology in Belgium. be performed for 243,313 reads with a mean length of 740 nucleotides. Reads with a q-score lower than 7 were filtered out, resulting in 179,015 remaining sequences mean length 816 nt for use in downstream analyses. Results of the sequencing run, including taxonomical classification and mapping of reads against PEDV and rotavirus A RVA reference genomes are shown in Fig. 1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database.", "1A . After 24 hours of sequencing, a total of 15,232 reads were classified as viral by sensitive tBLASTx comparison against a complete viral database. Of these, 39.3% n = 5,985 and 10.3% n = 1,564 were assigned to viral families comprising Porcine epidemic diarrhea virus family Coronaviridae and Rotavirus family Reoviridae, subfamily Sedoreovirinae , respectively. A fraction of the reads 29.3%, n = 4,468 were assigned to order Caudovirales. These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore.", "These reads originated from the lambda phage DNA used in a previous control run on the same flowcell. At 7.5 and 24.2 seconds after the start of sequencing, respectively, the first reads matching PEDV and RVA were translocated through a nanopore. Most reads were generated in the first twelve hours of sequencing and read accumulation was most exponential in the first three hours of sequencing Fig. 1B . PEDV and RVA sequences were extracted from the dataset and mapped against viral reference genes to calculate sequencing depths over time Fig. 1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X .", "1C . After one hour, sequencing depths were higher for PEDV 43.0X than for RVA 4.9 to 22.1X . High sequencing depths were acquired after three hours of sequencing for PEDV 103.5X and for most RVA gene segments 19.2 to 48.2X . De novo assembly was executed on the quality-filtered reads prior to identification tBLASTx to recover viral genomes. This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 .", "This resulted in the recovery of the almost complete PEDV genome and RVA gene segments with identities varying between 95 and 99% compared to the reference genes Table 1 . Higher assembly accuracies 97 to 99% were obtained when only the reads matching against rotavirus and PEDV were included for de novo assembly Table 1 . However, execution of de novo assembly prior to taxonomical classification tBLASTx reduced the time to identify entire viral genomes in the dataset. Virome composition of a young diarrheic piglet using nanopore sequencing. A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses.", "A total of 30,088 reads were generated by sequencing the diarrheic fecal sample for three hours. Of these, 25,466 reads q-score >7, mean read length 653 nt were used for further analyses. Different methods were used to compare the reads against a viral database using the HPC cluster of Ghent University and results are shown in Fig. 2 . Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx.", "Comparison against a complete viral database resulted in the detection of 6,781 to 8,677 potential viral reads, depending on the BLAST settings. BLASTn resulted in rapid taxonomical identification of reads at almost similar sensitivity compared to tBLASTx. However, there was a very high difference between wall times on the HPC cluster, with only 26 seconds of analysis time for BLASTn, versus almost 24 hours for tBLASTx. The majority of sequences were assigned to bacteriophages within the order Caudovirales and families Siphoviridae n = 3,213 to 4,163 reads , Podoviridae n = 2,506 to 3,002 reads and Myoviridae n = 912 to 1,202 reads . A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig.", "A de novo assembly was executed on the basecalled, quality filtered reads and the resulting contigs were used as input material for VirSorter analysis. Nineteen contigs were classified as sure n = 4; category 1 , somewhat sure =14; category 2 and not so sure =1; category 3 to be phage-like contigs Fig. 2B . Comparison of these contigs against the GenBank database using BLAST allowed classification into four different groups. Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown .", "Ten contigs showed moderate to high nucleotide similarities to the Bacteroides phage B124-14, suggesting that they all belonged to one phage genome. This was also supported by the fact that all these contigs mapped nicely distributed across the reference genome of Bacteroides phage B124-14 data not shown . The longest contig with a size of 39,069 nucleotides, together with four other contigs showed similarities 95% nt identity to different Escherichia phages. As they also mapped nicely distributed across the reference genome of Escherichia phage vB_EcoP_PhAPEC7, it seems that they must also belong to one phage genome data not shown . Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome.", "Two contigs showed poor similarity to both the Enterococcus phage vB_EfaS_IME_196, isolated from hospital sewage in China from an Enterococcus faecalis strain, and the Enterococcus hirae bacterial genome. The latter might be a prophage inserted in the bacterial genome. Interestingly, three contigs were identified for which no similarities were found with existing viruses in GenBank, but contig 0105 mapped to the reference genome of the Enterococcus phage vB_EfaS_IME196 data not shown . These might be novel phages or divergent variants from existing phages present in GenBank. Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae.", "Three eukaryotic porcine viruses, porcine kobuvirus n = 18 to 22 reads , enterovirus G n = 5 to 9 reads and astrovirus n = 4 reads were found at much lower abundancies. The genera Kobuvirus and Enterovirus belong to the family Picornaviridae, whereas the genus Mamastrovirus belongs to the family Astroviridae. Kobuvirus reads were mapped against a European reference strain S-1/HUN/2007/Hungary, as shown in Fig. 2C . However, full-genome coverage at high sequencing depth was not obtained. Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated.", "Shedding of porcine kobuvirus and rotaviruses in suckling piglets. The shedding of porcine kobuvirus, RVA and rotavirus C RVC was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated. The fecal shedding patterns of the different viruses and presence of diarrheic signs are shown in Fig. 3A . All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification .", "All piglets started shedding porcine kobuvirus at the end of the first week after parturition. In two piglets A and D the shedding was sustained and lasted for at least 2 weeks above the limit of quantification . Peak shedding titers of the porcine kobuvirus varied between 6.42 and 7.01 log 10 copies/swab, which is generally lower than peak shedding observed for typical enteric viruses such as rotavirus and PEDV. Moreover, the peak of shedding was not related to diarrheic episodes, questioning the role of this virus in the pathogenesis of diarrhea on the farm. Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed.", "Diarrheic signs were only noticed in two piglets A and B . In piglet B, an association between high RVC shedding and diarrheic episodes was observed. In contrast, there was no direct association between peak shedding of kobuvirus and diarrheic episodes. Interestingly, a peak in kobuvirus shedding was observed in piglet C at day 11 post-farrowing. This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine.", "This animal died shortly hereafter, but it was unclear if this can be attributable to the kobuvirus infection. Acute RVA shedding was observed at the end of the suckling period in three of five piglets, even though all sows were vaccinated before farrowing using a bovine inactivated rotavirus vaccine. Retrospective analysis of porcine kobuviruses shedding in Belgian diarrheic suckling pigs and phylogenetic analysis. A total of 44 diarrheic fecal samples collected in 2014 were screened for the presence of kobuvirus using the new RT-qPCR. Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification.", "Of these, 25 samples 56.8% tested positive and 18 samples showed quantifiable viral loads 4.31 to 6.83 log 10 copies/swab . Seven samples were positive, but viral loads were too low to allow accurate quantification. The presence of RVA and RVC had been quantitatively assessed in these samples in a previous study and the occurrence of co-infections between rotaviruses and kobuvirus is shown in Fig. 3B 25 . Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed.", "Kobuvirus was found in equal ratios in rotavirus-negative and -positive samples. Twelve samples contained a single rotavirus infection with a high RVA load and in four of these, a high kobuvirus load 5.16 to 5.42 log 10 copies/g was observed. A single RVC infection was found in seven samples and in four of these tested positive for kobuvirus at high loads 4.31 to 5.59 log 10 copies/g . A dual RVA/RVC infection was seen in two samples, but neither contained quantifiable kobuvirus loads. Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% .", "Many n = 10 of the rotavirus-negative samples contained high kobuvirus loads. Strain 17V079 showed high similarity to other Belgian porcine kobuvirus isolates from 2014 92.1 to 94.0% nucleotide sequence identity and the Hungarian reference strain S-1/Hun/2017 93.4% . Furthermore, there was a high level of genetic variability between the 2014 Belgian porcine kobuvirus isolates, with nucleotide sequence identities ranging between 90.1 and 97.2%. A phylogenetic analysis, using the 3D gene of 17V079 and twelve Belgian isolates from 2014 Fig. 3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools.", "3C , shows the Belgian strains clustering between strains from different geographical locations. Prevention and treatment of enteric disease problems in young piglets is frequently hampered by a lack of diagnostic tools. Veterinarians are restricted to a short list of known viruses, bacteria, parasites and management factors to define a differential diagnosis. Only the most likely cause s of the disease will be diagnostically investigated, often leading to negative, inconclusive or incomplete results. However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample.", "However, metagenomics studies have indicated the existence of viral enteric disease complexes, potentially involving multiple known and novel viruses 3, 4, 6, 7, 9, 10 . Detection of nucleic acids from pathogens using NGS-based metagenomics approaches is a partial solution to diagnostic testing problems and can provide a complete readout of viruses and other pathogens present in a sample. However, most NGS platforms require large investments and processing of the reads can only start at the end of the sequencing run. Viral metagenomics also requires extensive laboratory preparations, including centrifugation, filtration and nuclease treatment to discard bacterial and host nucleic acids that make up to the bulk of all nucleic acids present 13 . Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss.", "Furthermore, the amount of viral nucleic acids in a sample is very low, requiring targeted or random amplification of these genomes before NGS analysis. Amplification may induce bias and hampers the development of a fast diagnostic pipelines due to considerable time loss. All these factors lead to a long turnover time between sample collection and diagnostic reporting. The third-generation sequencing device MinION ONT , holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all DNA/RNA in a sample, theoretically without needing pre-amplification of viral nucleic acids. It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets.", "It was the aim of the present study to evaluate this technology for use as a rapid tool for porcine viral enteric disease complex identification, without the conduction of viral nucleic acid amplification. In a first experiment, cell culture-grown PEDV and RVA, known to induce diarrhea in young pigs, were pooled at high loads mimicking shedding quantities in diarrheic piglets. Sequencing of this pooled sample with the MinION resulted in rapid identification of both viruses. Real-time analysis of the sequencing reads was not conducted, but is achievable as previously demonstrated by Greninger and colleagues using the SURPI analysis pipeline for rapid identification of human viruses from different clinical matrices 19 . Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X .", "Interestingly, the first reads matching PEDV and RVA were generated respectively after 7 and 24 seconds of sequencing. High sequencing depths 43.0X were acquired within one hour of sequencing for PEDV and within three hours for most of the eleven RVA gene segments 19.2-48.2X . Overall, higher sequencing depths were generated for PEDV that could indicate that sequencing of longer viral genomes is favored over smaller gene segments, as PEDV has a genome size of approximately 28 kb, and RVA gene segments are shorter 0.6 to 3.3 kb . This bias might have been introduced during the ligation of the sequencing adapters to the viral nucleic acids. It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available.", "It can be hypothesized that adapters are more easily attached to longer DNA fragments, and bias should be avoided by standardization of viral nucleic acid input length. Rapid read generation allows flexible use of the sequencing platform and sequences can be read until enough genome information of the viruses of interest is available. While the technology can be useful for giving fast readouts of viruses <3 hours present in a sample, thorough validation, using well-defined virus stocks, spiking experiments in matrices e.g. feces and real clinical samples is necessary to make sure that all members of the porcine viral enteric disease complex are accurately being diagnosed. Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet.", "Furthermore, the accuracy of the technology needs further improvement, as error rates of contigs from de novo assemblies still ranged between 1 and 5%, hindering the precise analysis of subtle but important mutations in the viral genome. After the successful identification of the cell culture-grown viruses, the performance of the MinION was further explored by analyzing a diarrheic fecal sample of a one-week-old suckling piglet. Real-time PCR analyses were conducted for RVA, RVC, PEDV and TGEV. Enterococcus hirae was isolated at a private diagnostic laboratory, but this bacterial species is not considered a typical cause of diarrheic disease in pigs 26 . Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted.", "Viral metagenomics was conducted on this sample using the MinION and two different BLAST search algorithms were used to taxonomically identify the reads by comparing them against a complete viral database. Overall, tBLASTx with an e-value of 10 −3 was able to identify the most viral reads compared to other search options conducted. However, BLASTn search options also reached high sensitivity, but at much lower time cost: 26 seconds instead of almost 24 hours. For rapid read analysis and searching for closely related non-divergent viral sequences, BLASTn or another fast methodology should thus be preferentially used. However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079.", "However, tBLASTx might pick up more divergent or novel viruses, improving overall sensitivity. Three porcine viruses, including porcine kobuvirus, porcine mamastrovirus and enterovirus G, were identified in sample 17V079. Astro-and enteroviruses have been detected earlier in both diarrheic and non-diarrheic feces of Belgian pigs and in feces from pigs around the globe 9,10,27 . In a recent study from Thailand, the difference in prevalence of astrovirus in diarrheic 8.4% versus non-diarrheic 4.6% piglets less than 4-weeks-old was not statistically significant. Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 .", "Also other studies have shown that the role of porcine astrovirus in the pathogenesis of pig diarrhea is not completely clear 28 . In contrast, associations between diarrhea and human astrovirus infections have been made 29 . A recent study in 5 European countries Hungary, Spain, Germany, Austria and Sweden have indicated the widespread of porcine astroviruses in the swine population. A one hundred procent prevalence of astrovirus was found in diarrheic and non-diarrheic pigs from Austria and Spain. Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections.", "Porcine astroviruses have recently also been linked to outbreaks of neurological disorders in weaned piglets from Hungary, and in 5-week-old pigs and sows in the United States 30, 31 . The gut might be a hypothetical entry port for such neurological astrovirus infections. Enteroviruses have been more generally linked to neurological disorders in pigs, although they are commonly found in feces as well 11, 12, . In a study from Vietnam, no significant correlation was found between diarrhea status and presence of enterovirus G in feces 35 . The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads.", "The involvement of both astro-and enteroviruses in the pathogenesis of enteric disorders might be questioned here, but cannot be completely ruled out. Furthermore, while sensitive tBLASTx searches were used here, there is still a possibility that a completely novel virus might be present in the dark matter of the sequencing reads. However, reporting of a porcine kobuvirus in Belgian piglets with MinION is unique. In Belgium, kobuviruses had previously only been found in diarrheic samples of calves and young cattle in Belgium 36 . In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed.", "In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions. Similar kinetics were also analyzed for porcine rotavirus A and C. While suckling piglets started shedding porcine kobuvirus from one week of age, an association between peak viral shedding 6.42 to 7.01 log 10 copies/swab and diarrheic signs was not observed. In one pig, an association was made between diarrheic episodes and the peak of rotavirus C shedding, a well-known enteric pathogen 37, 38 . Very interestingly, kobuvirus fecal loads were typically lower than those reported of well-described enteric viruses of which the pathogenicity has been proven using piglet infection models, such as PEDV and rotavirus . Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 .", "Similar viral loads for porcine kobuvirus were also found in case 4.60 ± 1.76 copies/qPCR reaction and control pigs 4.79 ± 1.72 copies/qPCR reaction during a recent Danish study to evaluate the role of viruses in the pathogenesis of the new neonatal porcine diarrhea syndrome. The study demonstrated that kobuvirus, astrovirus, rotavirus A, porcine teschovirus, porcine norovirus and porcine coronaviruses were not involved in the pathogenesis of the syndrome 42 . The finding of low kobuvirus loads in feces casts doubt over the true enteric pathogenic tropism of the virus. Hypothetically, its replication is likely not distributed across the whole villus but limited to either enterocytes at the villus' tips or to immune cells present in the gut. The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission.", "The presence of kobuvirus RNA in serum has also been demonstrated in Hungarian pigs, but it was not known if the virus is also replicating in other organs 43 . Both the oro-fecal route and the feeding of milk to sucklings pigs could be involved in virus transmission. Highest rates of infection were observed in suckling piglets, compared to older pigs, in other countries . In our study, relatively long shedding of porcine kobuvirus was observed in three out of five animals, which may indicate that this virus may induce persistent infections. A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus.", "A 2011 Brazilian study demonstrated the presence of kobuvirus RNA in serum from 3-day-old piglets, which had disappeared by day 21, indicating viral clearance from the blood and excluding systemic persistence 45 . A complete lack of pathogenicity cannot be excluded, as porcine kobuviruses might play a role as a subclinically important virus. Such subclinical, yet immunosuppressive, properties have been attributed to the economically important swine pathogen porcine circovirus 47 . Of interest, one of the piglets died at the peak of kobuvirus shedding, although it was not clear if there was any causality between virus replication and the piglet's death. In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells.", "In vivo animal experiments in a model of neonatal, conventional kobuvirus-negative piglets should be conducted to elucidate the pathogenesis of porcine kobuviruses. Attempts were made to isolate the virus in different cell lines MA104, ST and SK , and peripheral blood mononuclear cells. There was no evidence of cytopathogenic effect after several days of incubation. Antibodies to visualize antigen expression were not available and therefore the possibility of replication without SCIenTIFIC REPORTS | . 8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available.", "8:9830 | DOI:10.1038/s41598-018-28180-9 evident cytopathogenic effect cannot be ruled out. Efforts will be made to isolate the virus in porcine primary enterocyte cultures, once available. To assess more broadly the prevalence of kobuvirus in the Belgian swine industry, a retrospective analysis of diarrheic samples from suckling piglets less than two weeks old was conducted. A high proportion 40.9% of the samples n = 44 contained quantifiable viral loads ranging between 4.31 to 6.83 log 10 copies/g feces. Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made.", "Viral loads found were thus comparable to the loads excreted by piglets in the longitudinal analysis and the above-mentioned study from Denmark, demonstrating the endemic presence of the virus in the Belgian swine population 42 . In the present study, non-diarrheic piglets were not included and therefore no association between kobuvirus prevalence and disease can be made. However, the prevalence of kobuvirus has been widely described in pigs from several European countries The Netherlands 16.7% , Slovakia 63.4% , Hungary 81.0% , Czech Republic 87.3% , Austria 46.2% , Italy 52.4% , Germany 54.5% and Sweden 45.0% , American countries The United States 21.9% and Brazil 53.0% , African countries Kenya 14.9% and Uganda 15.5% and Asian countries Thailand 99% , South Korea 52.1% and Vietnam 29.3% . In a small proportion of these studies, statistically significant associations between prevalence of kobuvirus and diarrhea in pigs were demonstrated, such as in Hungary 54.5% prevalence in healthy pigs vs 92.3% prevalence in diarrheic pigs , Spain 47.5% healthy vs 74.4% diarrheic , Brazil 41% vs 78.4% , Thailand 19.3% vs 84.5% and Vietnam 27.6% to 40.9% 35, 45, 46, 52 . Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%.", "Indeed, it is difficult to make correlations between prevalence of the virus and diarrhea, as the pathogenicity of the virus could be largely influenced by other factors such as co-infections with other enteric viruses, microbiota and management factors. Belgian isolates showed genetic moderate to high genetic variability, with nucleotide identities between 90.1 and 97.2%. Furthermore, they clustered diffusely between strains from different countries around the world, indicating that strains are not distinguishable based on their geographical origin. Because most 99% of the reads generated during sequencing of the fecal sample 17V079 matched bacteriophages upon analysis with BLAST, bacteriophages may have played an important role in the pathogenesis of the diarrheic disease. De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 .", "De novo assembled contigs were analyzed using VirSorter, a software package for mining viral signals from microbial genomic data. Such tools allow maximizing the possibility of detecting dsDNA phages 54 . Several contigs showed high similarities to the Bacteroides phage B124-14, found in municipal wastewater and human fecal samples. It was shown to be absent in 30 samples collected from different animal species, including pigs, and is therefore considered a human-specific phage 55, 56 . The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies.", "The finding of several contigs, genetically similar to phage B124 and likely belonging to one phage genome, indicates that this phage found in the pig fecal sample may also replicate in the microbiome of the young pig gut and not solely in humans. However, it is possible that the phage's replication ability in the pig's gut is age-dependent and that very young age groups were not sampled in previous studies. Interestingly, several of the contigs found also showed similarities to Escherichia phages. Two of the contigs were similar to Escherichia phages PhAPEC5 and PhAPEC7, isolated from Belgian rivers in the neighborhood of poultry houses and known to cause lytic infections in avian pathogenic Escherichia coli. Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 .", "Electron microscopic images of the phages PhAPEC5 and PhAPEC7 indicated that they belonged to the family Podoviridae 57 . Two other contigs were similar to two closely related Escherichia phages, St11Ph5 and G7C, found in sewage and horse feces, respectively 58 . Finally, one contig showed limited similarity to an Enterococcus phage, isolated from hospital sewage in China, while a last contig showed moderate similaraties to the bacterial Enterococcus hirae genome. This region may be a prophage, inserted in the bacterial genome. The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins.", "The phages found in this piglet may have reshaped the gut microbiota, allowing opportunistic bacteria such as Enterococcus hirae to proliferate and to start secreting toxins. It is also possible that a phage infection of bacteria in the pig's gut led to a stress status for these bacteria, prompting the secretion of toxins. The new neonatal diarrhea syndrome described above shows high similarities to the disease described in the case 17V079 and it may be that bacteriophages are involved in the pathogenesis of this syndrome. So far, the role of phages has not been considered in the pathogenesis of several enteric disorders, but given the high abundance here, it should be in future studies. It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant.", "It is clear that new technologies will change the way diagnostics are be performed in the near future. Pricing might currently be an aspect hampering high-troughput analysis of samples in swine veterinary medicine, but as the technology evolves fast, this might become very soon less relevant. Complete overviews of all viruses and other pathogens in a sample will be given in a single readout instead of requiring different diagnostic assays. However, care should be given to the interpretation of such results, as they should only be analyzed by trained veterinarians. Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml.", "Viruses. Porcine rotavirus A RVA strain RVA/Pig-tc/BEL/12R046/2012/G9P was isolated from a diarrheic piglet and grown for three successive passages in MA104 cells to an infectious virus titer of 10 7.8 CCID 50 / ml. The nucleotide sequences of the 11 gene segments of this strain were resolved earlier using Sanger sequencing GenBank accession numbers: KM82070 VP1 , KM820707 VP2 , KM827014 VP3 , KM820720 VP4 , KM820728 VP6 , KM820735 VP7 , KM820742 NSP1 , KM820672 NSP2 , KM820679 NSP3 , KM820686 NSP4 and KM820693 NSP5 59 . A porcine epidemic diarrhea virus strain PEDV, CV777 isolated in Belgium in the 1970s was adapted for growth in Vero cells in the 1980s 60 . In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets.", "In our Laboratory, the virus was grown to an infectious virus titer of 10 6.0 CCID 50 /ml GenBank accession number: AF353511 . Origin of a fecal sample from diarrheic suckling piglets. A diarrheic fecal sample was collected from a Belgian pig on a farm housing a total of 620 sows and using a 2-week batch-production system, with a weaning age of 23 days. Topigs Norsvin sows were crossed with Piétrain boars, producing 32. toxins Suiseng, Hipra . Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed.", "Rotavirus A vaccination was done off-label with an inactivated bovine rotavirus A vaccine Lactovac, Zoetis . Until recently, diarrheic problems were rarely present in suckling piglets and also very low mortality percentages 6.2-7.1% were observed. Since the spring of 2017, enteric disease started causing more severe problems accompanied with mortality on this farm, mainly in 7-days-old suckling piglets. A diarrheic fecal sample of such a piglet was investigated at a private diagnostic laboratory Dialab, Belsele, Belgium and labeled 17V079. No virological cause was found to explain the diarrheic problems on the farm. The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine .", "The only isolated bacterium was Enterococcus hirae. This bacterium was thereon added to the sow vaccination schedule inactivated autovaccine . No other pathogens were found in this sample. As the clinical picture hinted at a viral cause for the disease, the sample was sent to the Laboratory of Virology at the Faculty of Veterinary Medicine Ghent University for further analysis. The sample tested negative for RVA, RVC, PEDV and TGEV using in-house RT-qPCR assays 25, 61, 62 . Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids.", "Therefore, it was decided to perform a metagenomics analysis with MinION described in this study. Purification of viral nucleic acids. First, viral enrichment was done based on the NetoVIR protocol to obtain pure viral nucleic acids for sequencing library preparation 13 . MinION analyses of cell culture grown viruses RVA and PEDV were conducted at the Laboratory of Clinical Virology Rega Institute, KU Leuven , whereas the diarrheic fecal sample was analyzed at the Laboratory of Virology Faculty of Veterinary Medicine, Ghent University . RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses.", "RVA and PEDV stocks were centrifuged at 17,000 × g for 3 min. The supernatant of both suspensions was diluted to 6 log 10 CCID 50 /ml and 500 µl of each suspension was mixed to reach an equal concentration of both viruses. This mixture was filtered using a 0.8 µm polyethersulphone filter for 1 min at 17,000 × g, followed by a nuclease treatment for 2 hours at 37 °C to digest free nucleic acids in the suspension: 250 µl of the sample was added to 14 µl of home-made buffer 1 M Tris, 100 mM CaCl 2 and 30 mM MgCl 2 , pH 8 , 4 µl of Benzonase Nuclease Millipore and 2 µl Micrococcal Nuclease NEB as described earlier 13 . Fourteen microliters of EDTA were added to stop the reaction, followed by extraction of nucleic acids from the viral particles using the QIAamp Viral RNA Mini Kit Qiagen . The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications.", "The manufacturer's instructions were followed but no carrier RNA was added and elution was done in 30 µl of AVE to concentrate the viral nucleic acid extract. The diarrheic fecal sample 17V079 was processed similarly as the cell culture grown viruses, with some minor modifications. A 10% w/v suspension of the diarrhea was made in Minimum Essential Medium and centrifuged. The supernatant was filtered through a 0.45 µm syringe filter Sarstedt and treated with Benzonase Nuclease for 1 hour to speed up the diagnostic pipeline. Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl.", "Viral nucleic acids were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions without addition of carrier RNA. Elution was done in a volume of 50 µl. cDNA and second strand synthesis for nanopore sequencing. Nucleic acids were heated at 95 °C for 2 min and chilled on ice to resolve secondary RNA structures and to denature double-stranded RNA. Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water.", "Superscript IV Reverse Transcriptase ThermoScientific was used to generate cDNA. Ten microliters of template nucleic acids were mixed with 0.5 µl random hexamer primers Random Primer 6, New England Biolabs , 1 µl dNTP mix NEB and 2.5 µl nuclease-free water. Primer annealing was conducted at 65 °C for 5 min, after which 4 µl Superscript IV Reaction Buffer ThermoScientific , 1 µl dithiothreitol ThermoScientific and 1 µl SuperScript IV Reverse Transcriptase ThermoScientific were added in a total reaction volume of 20 µl. The reaction conditions were as follows: 23 °C for 10 min, 50 °C for 10 min, 80 °C for 10 min and an infinite hold step at 10 °C. A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume .", "A second strand of DNA was generated from single stranded c DNA molecules using the NEBNext Second Strand Synthesis Kit NEB . Twenty microliters cDNA reaction mixture were added to 10 µl NEBNext Second Strand Synthesis Reaction Buffer, 5 µl NEBNext Second Strand Synthesis Enzyme Mix and 45 µl nuclease-free water 80 µl total reaction volume . Isothermal amplification was done at 16 °C for 1 h and double-stranded nucleic acids were purified using 144 µl of magnetic AMPure XP Beads Beckman Coulter . Two washing steps with freshly prepared 70% ethanol were conducted before eluting in 52 µl nuclease-free water. Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters.", "Nanopore sequencing library preparation. A deoxyadenosine was ligated to the 3′-end of double-stranded nucleic acids to allow binding of complimentary sequencing adapters. Fifty microliters of un amplified DNA were mixed with 7 µl Ultra II End-Prep Reaction Buffer New England Biolabs and 3 µl Ultra II End-prep enzyme mix New England Biolabs , and incubated at 20 °C for 5 min and 65 °C for 5 min. Next, nucleic acids were purified using 60 µl AMPure XP Beads and eluted in 31 µl nuclease-free water. Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min.", "Sequencing adapters, provided with the Ligation Sequencing Kit 1D R9.4 SQK-LSK108, ONT , were ligated to the dA-tailed nucleic acids. End-prepped DNA 30 µl was mixed with 20 µl adapter mix AMX, ONT and 50 µl Blunt/TA Ligation Master Mix New England Biolabs in a total reaction volume of 100 µl and incubated at room temperature for 10 min. The sequencing library, containing double-stranded DNA with adapters ligated to the 3′ ends, was then purified using 40 µl AMPure XP beads. Two washing steps were conducted using 140 µl Adapter Bead Binding Buffer ABB, ONT before eluting in 15 µl of Elution Buffer ELB, ONT . EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT .", "EXP-LLB001, ONT , 12 µl adapted and tethered library and 12.5 µl nuclease-free water. Sequencing was done using the software programme MinKNOW software ONT . Bio-informatics analyses. Raw reads were produced by MinKNOW. Live basecalling was enabled for the first experiment using MinKNOW version 1.5.5. In the second experiment, basecalling was done after the sequencing run using Albacore version 1.2.5., ONT . Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted.", "Quality scores and read lengths were visualized using NanoPlot, followed by quality filtering with NanoFilt 63 . Reads with a q-score lower than 7 were omitted. Sequences were then analyzed using different BLAST methods including BLASTn and tBLASTx BLAST version 2.6.0; e-value cut-off 1e −3 -1e −10 to compare sensitivity and run-times to detect viral sequences among the reads. A complete viral database was composed of all virus sequences in GenBank taxonomy ID 10239, containing sequences up to 17th of September 2017 . The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses.", "The best hit lowest e-value was visualized using KronaTools 64 . Reads matching viruses were extracted using Seqtk and used in downstream analyses. GraphMap version 0.5.2 and Samtools version 1.6 were used for mapping of reads against reference sequences, while Canu 1.6 was used for de novo assembly of viral genomes . VirSorter was run using the 'Viromes' database to look for phages, with the Virome Decontamination Mode on to identify phage contigs 54 . Bio-informatics analyses were executed on a local computer cluster and the high-performance computing facilities of Ghent University. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene.", "The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Sanger sequencing of porcine kobuvirus polymerase gene. A porcine kobuvirus was discovered in the sample 17V079 using the MinION. The sequence of the 3D gene of porcine kobuvirus encodes the polymerase and is considered to be most conserved among different strains. The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 .", "The exact nucleotide sequence of this virus was verified using reverse transcripion polymerase chain reaction followed by Sanger sequencing, as low coverage was obtained with MinION. RT-PCR was executed using the OneStep RT-PCR Kit Qiagen with the newly designed primers Kobu_6049Fw and Kobu_7524Rv IDT DNA Technologies Table 2 . The RT-PCR reaction contained 5 µl 5 × Qiagen OneStep RT-PCR Buffer, 1 µl dNTPs, 3 µl of each primer 10 µm , 7 µl nuclease-free water, 1 µl OneStep RT-PCR enzyme mix and 5 µl template RNA or water total reaction volume of 25 µl . RT-PCR conditions were as follows: 50 °C for 30 min, 95 °C for 15 min, followed by 30 cycles of amplification 94 °C for 30 s, 50 °C for 30 s and 72° for 90 s and a final extension step at 72 °C for 1 min. Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light.", "Reactions were held at 10 °C prior to loading 5 µl PCR product with 1 µl of loading dye in a 1.5% agarose gel. Electrophoresis was conducted for 30 min at 100 V and PCR product was visualized by ethidium bromide staining and UV light. The amplicon was sent to GATC Constance, Germany for Sanger sequencing using an ABI 3730xl DNA Analyzer system. Quality control of the raw chromatograms was done using 4Peaks Nucleobytes BV, The Netherlands and BLASTn NCBI, United States . Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water.", "Specific RT-qPCR primers Table 2 for the porcine kobuvirus polymerase-encoding gene were designed using Primerquest and Oligoanalyzer IDT DNA Technologies to allow exact quantification in feces of piglets. Each RT-qPCR reaction consisted of 10 µl PrecisionPlus OneStep qRT-PCR Mastermix containing SYBR Green, ROX and an inert blue pipetting dye Primerdesign, Southampton, United Kingdom , 0.4 µl of each primer 200 nM and 6.2 µl nuclease-free water. Three microliters of template RNA or water were added to each tube containing 17 µl mastermix. A synthetic RNA positive control 175nt was generated by RT-PCR using the primers Kobu3D_qPCR +T7_Fw and Kobu3D_qPCR_Rv, followed by in vitro transcription of this PCR product using a T7 RNA polymerase. The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s .", "The positive control was measured using Nanodrop and used to setup a standard curve over a linear dynamic range LDR from six to one log 10 copies/reaction. Reaction conditions were as follows: 55 °C for 10 min and 95 °C for 2 min, followed by 40 cycles of denaturation 95 °C for 10 s and annealing 58 °C for 60 s . Detection of SYBR Green fluorescence was done at the end of each annealing phase. A melt curve analysis was executed to assess specificity of the amplicons generated. Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene.", "Each dilution point in the standard curve and each sample was tested in duplicates. Amplicons were analyzed once on an agarose gel to assess the correct length of the amplicon and Sanger sequencing was conducted to confirm the amplification of the partial porcine kobuvirus polymerase gene. Assays were valid if the efficiency over the LDR was between 90 and 110%, and R 2 of the standard curve replicates was >0.99. Quantification of the viral loads was possible if the Cq-values of two qPCR replicates fell within the LDR of the assay. Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable.", "Both replicates had to be positive for a sample to be considered as positive. If the Cq-values of specific amplicons have fallen behind the lowest point of the standard curve, the sample was considered positive but not quantifiable. Longitudinal investigation of kobuvirus and rotavirus shedding in suckling piglets. Upon characterization of the virome with the MinION, a longitudinal follow-up study was setup between August and September 2017. To warrant the health status of the pig stock, entrance to the farm was strictly regulated. Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer.", "Sampling was performed by the farmer. Detailed instructions and sampling materials were provided to the farmer. Sample collection in the longitudinal field study was done in agreement with the European legislation on animal experiments. Sample collection was approved by and done in accordance to the requirements of the Local Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering of Ghent University. One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period.", "One day after parturition of the sows, five litters were selected at random. Within each litter, one piglet was identified for longitudinal follow-up during the entire suckling period. A dry cotton rectal swab Copan was collected from each individual piglet at days 1, 5, 8, 11, 14, 17, 20 and 22 after birth. The swab was placed immediately in 2 ml of viral transport medium phosphate buffered saline containing 1000 U/ml penicillin Continental Pharma, Puurs, Belgium , 1 mg/ml streptomycin Certa, Braine l′Alleud, Belgium , 1 mg/ml gentamicin Life Technologies and 0.01% v/v Fungizone Bristol-Myers Squibb, Braine l′Alleud, Belgium in a sterile 15 ml falcon tube Sarstedt and stored at −20 °C. Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs.", "Every week, samples were collected from the farm and transported to the Laboratory of Virology. The farmer was asked to mark the tube of each sample for presence or absence of diarrheic signs. Upon arrival in the Laboratory of Virology, the samples were thawed and placed on a shaker for 30 min at 4 °C to release viral particles in the transport medium. Samples were extracted using the QIAamp Cador Pathogen Mini Kit according to the manufacturer's instructions and purified nucleic acids were eluted in 100 µl of AVE and stored at −70 °C until RT-qPCR analysis. RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 .", "RT-qPCR analysis was conducted, as described above, to quantify porcine kobuvirus genome copies per swab. Furthermore, RVA and RVC shedding was assessed using previously described in-house RT-qPCR assays 25, 61 . Belgian suckling pigs. Fecal samples n = 44 of diarrheic suckling piglets less than 2 weeks old were sent to a private laboratory by veterinarians Dialab, Belsele, Belgium for etiological diagnosis, as described earlier. These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 .", "These samples were collected in 2014 and stored at −70 °C in the laboratory. They had previously been evaluated for the presence of rotaviruses using RT-qPCR 25 . RNA extraction was conducted using the QIAamp Cador Pathogen Mini Kit Qiagen as described above and RT-qPCR was done to quantify the load of kobuvirus RNA copies. Samples with a quantifiable viral load were subjected to RT-PCR to amplify the 3D polymerase gene, after which Sanger sequencing was performed. The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 .", "The sequences encoding the polymerase of 11 Belgian porcine kobuvirus isolates were deposited into GenBank with accession numbers MH184664-MH184674. The sequences were used to conduct a multiple sequence alignment together with other porcine kobuvirus strains in MEGA 7 using the ClustalW plug-in 69 . A maximum-likelihood phylogenetic tree was constructed with RAxML using a general time reversible model with gamma distribution 20 cats, alpha: 0.121, LogLK = 14938.461 and heuristic branch swapping 70 . Tree editing was done using Affinity Designer Serif . Pairwise distances were calculated using the p-distance model in Mega with bootstrap values set at 500 replicates." ]

[ "Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection CDI , for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630.", "Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct putative targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds.", "These results identify for the first time direct putative targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds. Spo0A is the key regulator for sporulation . Most of our knowledge about the protein is based on work in Bacilli. Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay .", "Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay . The signaling cascade allows for the integration of environmental signals into the regulation of Spo0A dependent processes, including sporulation. The two functional domains, the N-terminal phosphorylation and dimerization domain receiver domain , and the C-terminal DNA binding effector domain are separated by a hinge region that is relatively poorly conserved . Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation .", "Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation . Extensive characterization of Spo0A targets has revealed a motif that represents a high affinity Spo0A binding site, the 0A box . The crystal structure of the DNA binding domain confirms specific and non-specific contacts between the protein and the consensus sequence . It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis , and solvent production in Clostridium acetobutylicum . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection CDI for recent reviews see .", "It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis , and solvent production in Clostridium acetobutylicum . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection CDI for recent reviews see . Though many people are asymptomatically colonized by C. difficile, the bacterium can cause serious health problems, such as pseudomembranous colitis and toxic megacolon, under the influence of risk factors such as age and antibiotic use. As a result, CDI was long regarded a nosocomial infection. Recently, however, an increase in the cases of community acquired CDI can be observed . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 BI/ NAP1 and 078 .", "Recently, however, an increase in the cases of community acquired CDI can be observed . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 BI/ NAP1 and 078 . Its main virulence factors are the major clostridial toxins A and B . In addition, certain strains of C. difficile, including ribotypes 027 and 078, additionally encode a binary toxin . C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow .", "C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow . There is limited knowledge about the regulation of sporulation in C. difficile. It has been reported that spo0A, as expected, is required for the formation of spores and the gene is required for persistence and transmission in mice . Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A .", "Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A . Similarly, it was reported that multiple orphan histidine kinases can phosphorylate Spo0A in C. acetobutylicum . Recently, it was reported that spo0A can be transcribed from a SigH-dependent promoter . It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes.", "It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes. Escherichia coli strains were routinely grown in Luria-Bertani broth or plates, supplemented with appropriate antibiotics. Chloramphenicol was used at a final concentration of 20 mg/mL for agar plates and 10 mg/mL for liquid cultures. Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL.", "Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL. Cloning was carried out using E. coli DH5a, overexpression was performed in E. coli Rosetta DE3 pLysS Novagen . C. difficile strains were grown in a glucose-free trypton-yeast based medium TTY; 3% w/v bactotrypton BD , 2% yeast extract Fluka , 0.1% w/v thioglycollate Sigma pH 7.4 , supplemented with 20 mg/mL of lincomycin when appropriate, or on CLO or TSS plates Biomerieux . All plasmids are listed in Table 1 . Primers obtained from Sigma Aldrich are listed in Text S1 and specific cycling conditions are available on request.", "All plasmids are listed in Table 1 . Primers obtained from Sigma Aldrich are listed in Text S1 and specific cycling conditions are available on request. Unless noted otherwise, PCR reactions were carried out using Pfu polymerase Fermentas according to the instructions of the manufacturer. Plasmid pWKS1251, for the overproduction of Spo0A-DBD carrying a C-terminal 66His-tag, was constructed as follows. A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO Invitrogen , yielding pWKS1247.", "A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO Invitrogen , yielding pWKS1247. This plasmid was digested with NdeI and XhoI, separated on a 1% agarose/0.56 TAE 20 mM Tris Acetate, 0.5 mM EDTA gel, the fragment corresponding to the DNA binding domain was recovered by gel-isolation using a GeneJET Gel Extraction kit, Fermentas and cloned into similarly digested pMF14 that had been gel-isolated in the same manner. The construct was verified by PCR, restriction analyses and DNA sequencing using primers oWKS-135 and oWKS-136 see below . Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO.", "Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO. The inserts, and in the case of the mutated PabrB promoters the presence of the desired point mutations in the consensus 0A box, were verified by DNA sequencing using primers oWKS-24 and oWKS-25 see below . Sequence grade plasmids were isolated using a Nucleospin Plasmid QuickPure kit Macherey Nagel according to the manufacturer's instructions, except that two lysis reactions were combined onto a single filter and eluted with 65uC prewarmed AE buffer. All constructs were sequenced using BigDye Terminator chemistry Invitrogen on an ABI3130 sequencer Perkin Elmer , according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix Invitrogen in a final volume of 20 mL.", "All constructs were sequenced using BigDye Terminator chemistry Invitrogen on an ABI3130 sequencer Perkin Elmer , according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix Invitrogen in a final volume of 20 mL. After thermocycling, DNA was precipitated and washed with 65% isopropanol, and dissolved in 12 mL HiDi formamid Invitrogen at 96uC for 2 mins and stored in the dark at 4uC until the sequencing run. Sequence analyses were performed in CloneManager Professional Suite 7 SciEd and Geneious version 5.6.2 Biomatters Ltd . Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta DE3 pLysS Novagen . Transformants were used to inoculate 25 mL of LB with appropriate antibiotics.", "Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta DE3 pLysS Novagen . Transformants were used to inoculate 25 mL of LB with appropriate antibiotics. After overnight incubation, the cells were 1:100 diluted in 500 mL fresh medium containing appropriate antibiotics. Protein production was induced with 1 mM IPTG at an OD600 of 0.7 and growth was continued for another three hours before harvesting. Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described .", "Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described . In short, cells were disrupted in 4 mL lysis buffer 2 mM PMSF, 10 mM imidazole, 5 mM beta-mercaptoethanol, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . Cleared cell lysates we incubated with 2 mL pre-equilibrated 50% TALON slurry Clontech in a final volume of 15 mL lysis buffer for 1 hr. The resin was allowed to settle on a Poly-Prep column BioRad and washed with 2 mL wash buffer 20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column identical to wash buffer but with 50, 100, 250 or 500 mM imidazole .", "The resin was allowed to settle on a Poly-Prep column BioRad and washed with 2 mL wash buffer 20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column identical to wash buffer but with 50, 100, 250 or 500 mM imidazole . The whole procedure was carried out at 4uC. Fractions were assayed for purity and yield and suitable fractions were dialysed against 26 1L dialysis buffer 50 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM DTT using Slide-A-Lyzer cassettes with a molecular weight cut-off of 3.5 kDa Pierce . Proteins were stored at 280uC in storage buffer identical to dialysis buffer but containing 20% glycerol . Protein concentrations were determined using Bradford reagent BioRad , according to the manufacturer's instructions.", "Proteins were stored at 280uC in storage buffer identical to dialysis buffer but containing 20% glycerol . Protein concentrations were determined using Bradford reagent BioRad , according to the manufacturer's instructions. DNA fragments for use in EMSA experiment were generated by PCR using GoTaq polymerase Promega and chromosomal DNA from B. subtilis JH642 Bacillus Genetic Stock Center 1A96; plasmids listed in Table 1 , or chromosomal DNA from C. difficile 630Derm as a template. Primers and specific cycling conditions for generation of the EMSA probes are listed in Text S1. DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS and a QIAExII kit Qiagen , according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase Invitrogen according to the instructions of the manufacturer.", "DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS and a QIAExII kit Qiagen , according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase Invitrogen according to the instructions of the manufacturer. Specific activity was determined on a LS6000 scintillation counter Beckman . EMSA conditions were based on previous studies . In short, binding reactions were carried out in binding buffer 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol in the presence of 200 mg/mL bovine serum albumin NEB and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer.", "In short, binding reactions were carried out in binding buffer 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol in the presence of 200 mg/mL bovine serum albumin NEB and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer. Electrophoresis was carried out for 120 min at 85 V. After vacuum drying the gels onto filter paper, they were imaged after overnight exposure on Phosphorimager screens on a Typhoon instrument GE Healthcare . The toxic effects of C. difficile culture supernatants on Vero cells a kind gift of Eric Snijder were determined as follows. Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium Dulbecco modified Eagle medium Lonza supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum , before applying them to a monolayer of Vero cells, and incubation was continued for another hour.", "Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium Dulbecco modified Eagle medium Lonza supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum , before applying them to a monolayer of Vero cells, and incubation was continued for another hour. As a positive control, 50 mL 1:10 diluted purified toxin Techlab was added to the cells. To determine if observed cytotoxic effects were specific for the large clostridial toxins, commercially available anti-toxin against TcdA and TcdB Techlab was added to 10-fold diluted bacterial supernatant for 60 min prior to incubation on the Vero cells. Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects cell rounding were observed. Statistical significance was evaluated with an independent sample t-test.", "Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects cell rounding were observed. Statistical significance was evaluated with an independent sample t-test. Immunization of mice with full length C. difficile Spo0A-6xHis was kindly performed at the Welcome Trust Sanger Institute Hinxton, UK . Cells from 1 mL of C. difficile culture were collected by centrifugation for 1 min at 14000 rpm in a table top centrifuge and resuspended in 200 mL resuspension buffer 10 mM Tris HCl pH 8, 10 mM EDTA, 0.5 mg/mL lysozyme, 1 mM Pefabloc SC Roche . After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer 0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB was added, and samples were heated to 96uC for 5 mins. Total cell lysates amounts corrected for OD 600 were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride PVDF membrane.", "After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer 0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB was added, and samples were heated to 96uC for 5 mins. Total cell lysates amounts corrected for OD 600 were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride PVDF membrane. Membranes were blocked in PBST buffer phosphate buffered saline with 0.1% v/v Tween-20 containing 5% membrane blocking reagent Amersham Biosciences . To visualize Spo0A protein cleared polyclonal serum from a single mouse at a 1:3000 dilution was used, followed by either a goat-anti-mouse HRP-conjugated secondary antibody followed by ECL+ detection Amersham Bioscience , or a goatanti-mouse-biotin-conjugated secondary antibody Dako followed by a tertiary mouse-anti-biotin Cy3-conjugated antibody Jackson . Detection was done using on a Typhoon instrument GE Healthcare . Background corrected peak volumes were quantified using ImageQuant TL Amersham Biosciences .", "Detection was done using on a Typhoon instrument GE Healthcare . Background corrected peak volumes were quantified using ImageQuant TL Amersham Biosciences . Alignments of B. subtilis and C. difficile spo0A were made using ClustalW2 on the basis of the published genome sequences, Genbank accession numbers AL009126 and AM180355, respectively, and the 630Derm spo0A sequence as determined in this study. The sequence for spo0A of C. difficile strain 630Derm was deposited in Genbank accession no JX050222 . Consensus Spo0A boxes were identified using a Single string Search command in Genome2D , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel.", "Consensus Spo0A boxes were identified using a Single string Search command in Genome2D , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel. The results were manually inspected for those boxes within 500 bp upstream of a gene on the same strand. Figures for publication were prepared using ImageQuant TL Amersham Biosciences , Adobe Photoshop CS3 Adobe Systems Inc and Corel Graphics Suite X5 Corel Corporation . In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain DBD were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli Fig. 1A and purified to near homogeneity using metal affinity chromatography Fig.", "In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain DBD were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli Fig. 1A and purified to near homogeneity using metal affinity chromatography Fig. 1A ; lanes P . Full length protein was used to raise antibodies to detect Spo0A in total lysates of C. difficile strains, and the purified DNA binding domain was used in subsequent in vitro binding assays see below . We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells.", "We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells. We performed immunoblotting using polyclonal antibodies against C. difficile Spo0A on total lysates of wild type and spo0A mutant cells grown in a trypton-yeast based medium TTY . We found a clear signal of the size expected for full length Spo0A ,31 kDa as early as 3 hours post inoculation exponential growth phase , through transition phase 8 h as well as 24 and 48 hours post inoculation stationary growth phase Figure 1B; 630Derm . The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant Fig. 1B , CT::spo0A .", "The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant Fig. 1B , CT::spo0A . We obtained similar results in other media, such as the commonly used supplemented brain heart infusion broth BHIS; data not shown . To determine relative levels of Spo0A throughout growth, we performed an immunoblot experiment using fluorescent antibodies, which gives more quantitative information compared to the use of horseradish peroxidase conjugated antibodies in our hands. We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation Figure 1C . Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase.", "We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation Figure 1C . Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase. Spo0A of C. difficile Strain 630Derm Contains a 6aminoacid Duplication BLAST homology searches readily identify a homolog of the well-characterized B. subtilis Spo0A protein in C. difficile 630 CD1214 and previous work demonstrated that a spo0A mutant an insertional inactivation of cd1214 -as expected -no longer forms spores . In silico analyses suggest a similar secondary structure for both proteins Fig. 2A , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region . We compared the sequence of CD1214 obtained from our lab strain 630Derm to that of the published C. difficile 630 genome .", "2A , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region . We compared the sequence of CD1214 obtained from our lab strain 630Derm to that of the published C. difficile 630 genome . Strain 630Derm is a spontaneous erythromycin sensitive strain, which is commonly used in mutagenesis studies and was obtained by serial passaging of strain 630 . The 630Derm spo0A sequence Genbank accession no JX050222 was derived from the expression plasmids constructed for this study, and confirmed in a whole genome sequence of strain 630Derm generated in our lab data not shown . We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid NVGNIE duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation hinge , flanking the highly conserved DNA binding domain Fig.", "We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid NVGNIE duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation hinge , flanking the highly conserved DNA binding domain Fig. 2A and B . We verified the absence of this duplication in strain 630 by PCR Fig. 2C as well as sequencing from the chromosomal DNA of C. difficile 630 data not shown , to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 to which 630 and 630Derm belong by PCR, but the duplication was found to be unique to 630Derm among the isolates tested data not shown .", "2C as well as sequencing from the chromosomal DNA of C. difficile 630 data not shown , to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 to which 630 and 630Derm belong by PCR, but the duplication was found to be unique to 630Derm among the isolates tested data not shown . C. difficile Spo0A-DBD Shows Similar Specificity as B. subtilis Spo0A-DBD Next, we examined the conservation of the DNA binding domain of Spo0A Spo0A-DBD between B. subtilis and C. difficile. In B. subtilis amino acid residues contacting the backbone of the DNA and interacting with specific residues of the Spo0A binding sequence have been defined . We found that all these residues were conserved in the C. difficile protein sequence Fig. 2B , indicating that the protein likely recognizes a similar motif.", "We found that all these residues were conserved in the C. difficile protein sequence Fig. 2B , indicating that the protein likely recognizes a similar motif. DNA binding by full length Spo0A in B. subtilis requires phosphorylation dependent dimerization . However, it was shown that the isolated DBD is capable of binding to legitimate targets of the full length protein . Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene PabrB of B. subtilis.", "Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene PabrB of B. subtilis. PabrB is commonly used as a high-affinity control in binding assays with the B. subtilis Spo0A or Spo0A-DBD protein . It is noteworthy that we failed to identify a homolog of abrB in C. difficile using BLAST, indicating that potential indirect regulation by Spo0A cannot occur through abrB in C. difficile as it does in B. subtilis. We found that C. difficile Spo0A-DBD bound with high affinity to PabrB Fig. 2D and E .", "We found that C. difficile Spo0A-DBD bound with high affinity to PabrB Fig. 2D and E . We performed electrophoretic mobility shift assays EMSAs using radiolabeled PabrB and increasing amounts of purified C. difficile Spo0A-DBD that was purified using a C-terminal 66His-tag. The addition of protein leads to a dose-dependent retardation of the DNA fragment with an apparent K D of ,50 nM. In the same range of protein concentrations, no binding was observed for a negative control a DNA fragment of B. subtilis citG Fig. 2E , suggesting that binding was specific for the abrB promoter region. B. subtilis Spo0A recognizes a distinct sequence 0A box , that is characterized by a 7 bp core motif TGTCGAA .", "2E , suggesting that binding was specific for the abrB promoter region. B. subtilis Spo0A recognizes a distinct sequence 0A box , that is characterized by a 7 bp core motif TGTCGAA . Structural studies have revealed that the protein makes specific contacts with the G at position 2 G2 , and the C at position 4 C4 and 5 G5 of this motif . We introduced G2A, C4A, G5A, G2A/C4A and C4A/G5A mutations in the perfect consensus core 0A-box present in PabrB. We found that the affinity of C. difficile Spo0A for these mutated PabrB fragments was highly reduced Fig. 2E . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence.", "2E . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence. For the single point mutations in the DNA, the affinity decreased ,10-fold. There did not seem to be an additive effect of a second point mutation for the two combinations tested. None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other non-consensus 0A boxes in the abrB promoter . Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD.", "None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other non-consensus 0A boxes in the abrB promoter . Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD. Value for Binding by C. difficile Spo0A-DBD Above, we have established that the Spo0A-DBD of C. difficile is highly homologous to that of the B. subtilis Spo0A protein, and that the proteins recognize a similar consensus sequence Fig. 2 . Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A. We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D .", "Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A. We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D . Such an analysis revealed the presence of 102 matching motifs, of which 45 were located within 500 bp of the initiating ATG of an open reading frame on the same strand see Table S1 . Our attention was drawn to spo0A and sigH, as these two genes were previously found to be regulated by Spo0A in B. subtilis and/or play important roles in sporulation 3, . We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH. We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames.", "We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH. We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames. We found that the addition of Spo0A-DBD to the reactions caused retardation of the spo0A and sigH DNA fragments Fig. 3A , but not of a spoVG fragment which did not contain a consensus 0A box Fig. 3B . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA.", "3B . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA. Moreover, the presence of multiple shifted species could indicate the presence of more than one strong binding site. These results establish that spo0A and sigH are likely legitimate targets of Spo0A in C. difficile, and confirm that spoVG is not, in line with results obtained in B. subtilis . We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon.", "We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon. This positioning is similar to that observed for spo0A . and sigH .. We confirmed in vitro binding of the C. difficile Spo0A-DBD to the promoter regions of lplA and ssuA. We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene CD1654; box at 267 and the ssuA gene CD1484; box at 282 Fig.", "We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene CD1654; box at 267 and the ssuA gene CD1484; box at 282 Fig. 3A . The lplA gene encodes a predicted lipoate-protein ligase, and ssuA is annotated as an aliphatic sulfonates ABC transporter; to our knowledge, neither of these have been directly implicated in sporulation or have found to be targets for Spo0A in other organisms. Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile. It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway .", "Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile. It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway . However, the in silico identification of upstream regions with a consensus Spo0A binding site did not point to any of the early sporulation genes downstream of spo0A itself as direct targets of Spo0A. This is likely the result of variations in the 0A-box in these promoters that were disregarded in the box search. In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A such as spoIIAA and spoIIE do not contain a 100% match to the core motif, but rather one or more near-consensus boxes . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above Fig.", "In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A such as spoIIAA and spoIIE do not contain a 100% match to the core motif, but rather one or more near-consensus boxes . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above Fig. 3B . For spoIIAA encoding an antianti sigma-factor and spoIIE encoding a serine phosphatase , we observed a low intensity shifted species at concentrations as low as 150 nM. For spoIIGA encoding a sporulation specific protease we observed the shifted species only at higher concentrations of protein .200 nM . The negative control spoVG did not demonstrate binding of Spo0A-DBD at these concentrations.", "For spoIIGA encoding a sporulation specific protease we observed the shifted species only at higher concentrations of protein .200 nM . The negative control spoVG did not demonstrate binding of Spo0A-DBD at these concentrations. Moreover, the shift we observed was reversible using unlabeled DNA containing a high affinity binding site, but not using unlabeled DNA that lacked such a site Figure S1B-D . Therefore, we consider the binding to spoIIAA, spoIIE and spoIIGA genes to be specific, despite the fact that increasing the amount of protein did not seem to cause a significant increase in the amount of DNA in the complex. Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions. C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells .", "Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions. C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells . Considering the absence of a homolog of the abrB repressor, direct binding of Spo0A and concomitant activation of toxin gene transcription is a likely mechanism through which this could occur. We found evidence for direct binding of Spo0A-DBD to the region upstream of tcdB, encoding one of the major clostridial toxin genes, and possibly tcdC, but this did not seem to result in lower toxin levels in our hands. We performed EMSAs using DNA upstream of tcdR encoding a sigma factor responsible for the activation of toxin gene transcription , tcdB encoding toxin B , tcdA encoding toxin A . In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE encoding a holin-like protein and tcdC encoding a putative negative regulator of toxin production , even though this regulator does not have a significant effect on toxin levels under the conditions we used .", "We performed EMSAs using DNA upstream of tcdR encoding a sigma factor responsible for the activation of toxin gene transcription , tcdB encoding toxin B , tcdA encoding toxin A . In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE encoding a holin-like protein and tcdC encoding a putative negative regulator of toxin production , even though this regulator does not have a significant effect on toxin levels under the conditions we used . Of the regions tested, we only observed a clear shifted species, indicative of Spo0A binding, for tcdB Figure 4A ; the shifted species in our EMSA assay was reversed by the addition of unlabeled DNA containing a high affinity binding site, but not by DNA lacking such a site Figure S1E . For tcdC, some smearing was observed at all concentrations of proteins tested Figure 4A , and there did not seem to be a clear effect of the addition of unlabeled DNA fragments Figure S1F . The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG. We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested.", "The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG. We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested. We found no lower toxicity towards Vero cells of culture supernatants derived from spo0A mutant cells compared to wild type. We grew three independent biological replicates of a wild type 630Derm or Clostron-generated spo0A mutant CT::spo0A -a kind gift of the Minton lab in glucose-free TTY medium. We harvested culture supernatant at late-exponential phase approximately 7 hours post inoculation , the transition phase between exponential and stationary growth phase approximately 9 hours post inoculation , as well as two time points in stationary phase 24 and 48 hours post inoculation and determined the toxin endpoint titres see Materials and Methods . In contrast to previous findings, we observed a small #4-fold increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant p.0.05, independent sample t-test .", "We harvested culture supernatant at late-exponential phase approximately 7 hours post inoculation , the transition phase between exponential and stationary growth phase approximately 9 hours post inoculation , as well as two time points in stationary phase 24 and 48 hours post inoculation and determined the toxin endpoint titres see Materials and Methods . In contrast to previous findings, we observed a small #4-fold increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant p.0.05, independent sample t-test . In other medium BHIS , we observed no differences at all data not shown . We conclude that Spo0A does not positively affect toxin production in C. difficile 630Derm and the in vivo relevance of the binding to regions upstream of tcdB and/or tcdC is therefore limited under our experimental conditions. The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box .", "The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box . Depending on the analysis, this motif is flanked by one or more adenine or thymine residues . Interestingly, many target genes do not harbor a perfect match to this consensus sequence, but rather contain one or more degenerate motifs. The differences in these motifs may reflect different promoter architectures e.g. AT content , modes of action e.g.", "The differences in these motifs may reflect different promoter architectures e.g. AT content , modes of action e.g. activation or repression or levels of regulation. Spo0A genes in B. subtilis can be divided in different classes that respond to different levels of phosphorylated Spo0A . For C. difficile, we conclude that the Spo0A protein likely recognizes a motif that is similar to the B. subtilis Spo0A box on the basis of four lines of evidence; 1. All DNA binding/contacting residues are conserved Fig. 2B , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A Fig. 2D , 3.", "2B , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A Fig. 2D , 3. Mutagenesis of key residues in the B. subtilis Spo0A box reduces affinity of C. difficile Spo0A for DNA Fig. 2E and 4. A B. subtilis Spo0A box has predictive value for DNA binding by C. difficile Spo0A Fig. 3A . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein.", "3A . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein. It is likely that differences do exist between the preferred binding sites for both proteins that will be evident when a comprehensive analysis is performed of in vivo DNA binding of C. difficile Spo0A; based on the limited data set of this study, a MEME analysis already suggests possible differences in the extended Spo0A motif W.K. Smits, unpublished observations . These differences may relate to the much higher AT content of C. difficile compared to B. subtilis 71 vs. 56.5%, respectively , or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation for review see ref .", "These differences may relate to the much higher AT content of C. difficile compared to B. subtilis 71 vs. 56.5%, respectively , or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation for review see ref . The activation of Spo0A is controlled by a multi-component phosphorelay that can integrate environmental cues and ensures a gradual increase in the level of phosphorylated Spo0A in the cell . In addition, the transcription of the spo0A gene is controlled by multiple feedback loops. For instance, Spo0A regulates its own transcription by binding to the spo0A promoter , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter . In C. difficile, there are some interesting differences and similarities in the regulatory pathways.", "For instance, Spo0A regulates its own transcription by binding to the spo0A promoter , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter . In C. difficile, there are some interesting differences and similarities in the regulatory pathways. Most notably, there seems to be no phosphorelay and the phosphorylation state of Spo0A is supposedly controlled by orphan histidine kinases . The transcription of spo0A in C. difficile is under control of the transition state sigma factor Sigma H , as it is in B. subtilis . Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile Fig. 3A , raising the possibility of auto-regulation of spo0A.", "Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile Fig. 3A , raising the possibility of auto-regulation of spo0A. The putative direct regulation of sigH by Spo0A may reflect that the C. difficile genome does not harbor a homolog of the pleiotropic regulator AbrB, which is responsible for the Spo0A-dependent regulation of sigH in B. subtilis . Consistent with a model in which spo0A is positively autoregulated, we noted a sharp increase in the levels of Spo0A as cells approach the stationary growth phase Figure 1C . Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA Fig. 3B .", "Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA Fig. 3B . All these observations are consistent with direct regulation of these genes by Spo0A in other organisms , and the conservation of the sporulation pathway . Though Spo0A is the key regulator for sporulation in Firmicutes, it regulates numerous other processes in various bacteria. In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology 10, . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB.", "In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology 10, . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB. Additionally, transcription of abrB responds already to low levels of Spo0A,P . As a result these effects are detectable in late-exponential and early stationary phase, as some Spo0A is present throughout growth in B. subtilis cells. Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect.", "Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect. In Clostridium acetobutylicum and C. beijerinckii, Spo0A is a direct regulator of solvent formation, as well as sporulation . It seems therefore conceivable that Spo0A in C. difficile also affects aspects of metabolism. In this respect, it is important to note that also in C. difficile Spo0A is detectable from early exponential growth phase on Figure 1B . We observed direct binding of C. difficile Spo0A to the promoter region of sigH Fig. 3A .", "We observed direct binding of C. difficile Spo0A to the promoter region of sigH Fig. 3A . This gene encodes the key sigma factor for the transition phase, and regulates processes outside sporulation as well . Moreover, we found significant levels of Spo0A from early stationary phase on Fig. 1B and unpublished observations , indicating the regulatory actions of Spo0A need not be limited to stationary phase in C. difficile. In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA Fig. 3A .", "In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA Fig. 3A . The presence of a putative Spo0A binding site upstream of these genes, as well as the spacing compared to the start codon, is conserved in the problematic Stoke-Mandeville strain R20291 , a member of PCR ribotype 27. This could indicate that these aspects of regulation by Spo0A are conserved in multiple strains of C. difficile. It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed.", "It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed. In order to distinguish direct from indirect effects, in vivo binding profiles of Spo0A should be performed. The antibodies generated for this study should prove to be useful for this type of experiments. Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control .", "Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control . Similarly, the production of the emetic toxin cereulide in B. cereus is greatly repressed in a spo0A mutant, in an AbrB-dependent manner . In contrast, Spo0A directly represses the expression of the cry toxin genes in B. thuringiensis and a spo0A mutant is therefore a hyper-producer of the insecticidal crystal protein . In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A and also the production of enterotoxin in this organism seems to be indirectly dependent on sporulation . In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A TcdA , both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B TcdB .", "In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A and also the production of enterotoxin in this organism seems to be indirectly dependent on sporulation . In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A TcdA , both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B TcdB . Our in vitro binding data indicate a potential binding site for Spo0A upstream of tcdB and possibly tcdC Fig. 4A . However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant a kind gift of the Minton lab; did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type #2fold in exponential phase cells up to 4-fold in late-stationary phase cells .", "However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant a kind gift of the Minton lab; did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type #2fold in exponential phase cells up to 4-fold in late-stationary phase cells . The small, and not significant, differences in toxin levels in our experiments might be attributed to differences in the susceptibility of cells for lysis rather than the production of toxin, but could also indicate a negative regulatory effect of Spo0A on toxin production. In support of the latter hypothesis, it was recently reported that a spo0A mutant of C. difficile strain R20291 a PCR ribotypes 027/BI/NAP1 epidemic strain demonstrates ,10fold higher toxin levels than its isogenic wild type 30 h post inoculation, and is significantly more virulent in a mouse model of disease . The differences between Underwood et al on the one hand and our study as well as the study of Deakin and coworkers on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously data not shown .", "The differences between Underwood et al on the one hand and our study as well as the study of Deakin and coworkers on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously data not shown . Alternatively, the differences could indicate integration of the group II intron at more than one location in the chromosome in the strain used in Underwood et al . In the absence of a complementation experiment and/or Southern blot data, this remains to be established. In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays.", "In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays. In the present study we have for the first time demonstrated direct binding of the DNA binding domain of C. difficile Spo0A to putative target DNA. This work has revealed that aspects of Spo0A binding are conserved between Bacillus and C. difficile 0A box, possible auto-regulation and binding to early sporulation promoters , whereas others are not the absence of abrB as a direct target in C. difficile, binding to DNA upstream of lplA, ssuA . The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo.", "The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo. The identification of genes affected by Spo0A in C. difficile may shed light on the role of the protein in virulence and pathogenesis of this organism. Figure S1 Specificity controls for binding by Spo0A-DBD-his6. Arrows indicate the position of shifted species DNA:protein complexes . Titrations with PCR fragments of PabrB containing a high affinity binding site and PtcdA lacking such a site correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A.", "Titrations with PCR fragments of PabrB containing a high affinity binding site and PtcdA lacking such a site correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A. Comparison of binding of Spo0A-DBD-his6, Spo0A-his6 and CD2195-his6 binding to the upstream region of spoIIAA. B. Binding of Spo0A-DBD-his6 to the upstream region of spoIIAA is reversed by the addition of PabrB, but not by the addition of PtcdA . C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA.", "C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA. E. Binding of Spo0A-DBD-his6 to the upstream region of tcdB is reversed by the addition of PabrB, but not by the addition of PtcdA. F. Binding of Spo0A-DBD-his6 to the upstream region of tcdC is not or moderately affected by the addition of PabrB and/or PtcdA. TIF Text S1 Oligonucleotides used in this study and PCR cycling conditions for the EMSA probes. PDF" ]

[ "Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection CDI , for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630.", "Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct putative targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds.", "These results identify for the first time direct putative targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds. Spo0A is the key regulator for sporulation . Most of our knowledge about the protein is based on work in Bacilli. Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay .", "Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay . The signaling cascade allows for the integration of environmental signals into the regulation of Spo0A dependent processes, including sporulation. The two functional domains, the N-terminal phosphorylation and dimerization domain receiver domain , and the C-terminal DNA binding effector domain are separated by a hinge region that is relatively poorly conserved . Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation .", "Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation . Extensive characterization of Spo0A targets has revealed a motif that represents a high affinity Spo0A binding site, the 0A box . The crystal structure of the DNA binding domain confirms specific and non-specific contacts between the protein and the consensus sequence . It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis , and solvent production in Clostridium acetobutylicum . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection CDI for recent reviews see .", "It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis , and solvent production in Clostridium acetobutylicum . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection CDI for recent reviews see . Though many people are asymptomatically colonized by C. difficile, the bacterium can cause serious health problems, such as pseudomembranous colitis and toxic megacolon, under the influence of risk factors such as age and antibiotic use. As a result, CDI was long regarded a nosocomial infection. Recently, however, an increase in the cases of community acquired CDI can be observed . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 BI/ NAP1 and 078 .", "Recently, however, an increase in the cases of community acquired CDI can be observed . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 BI/ NAP1 and 078 . Its main virulence factors are the major clostridial toxins A and B . In addition, certain strains of C. difficile, including ribotypes 027 and 078, additionally encode a binary toxin . C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow .", "C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow . There is limited knowledge about the regulation of sporulation in C. difficile. It has been reported that spo0A, as expected, is required for the formation of spores and the gene is required for persistence and transmission in mice . Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A .", "Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A . Similarly, it was reported that multiple orphan histidine kinases can phosphorylate Spo0A in C. acetobutylicum . Recently, it was reported that spo0A can be transcribed from a SigH-dependent promoter . It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes.", "It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes. Escherichia coli strains were routinely grown in Luria-Bertani broth or plates, supplemented with appropriate antibiotics. Chloramphenicol was used at a final concentration of 20 mg/mL for agar plates and 10 mg/mL for liquid cultures. Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL.", "Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL. Cloning was carried out using E. coli DH5a, overexpression was performed in E. coli Rosetta DE3 pLysS Novagen . C. difficile strains were grown in a glucose-free trypton-yeast based medium TTY; 3% w/v bactotrypton BD , 2% yeast extract Fluka , 0.1% w/v thioglycollate Sigma pH 7.4 , supplemented with 20 mg/mL of lincomycin when appropriate, or on CLO or TSS plates Biomerieux . All plasmids are listed in Table 1 . Primers obtained from Sigma Aldrich are listed in Text S1 and specific cycling conditions are available on request.", "All plasmids are listed in Table 1 . Primers obtained from Sigma Aldrich are listed in Text S1 and specific cycling conditions are available on request. Unless noted otherwise, PCR reactions were carried out using Pfu polymerase Fermentas according to the instructions of the manufacturer. Plasmid pWKS1251, for the overproduction of Spo0A-DBD carrying a C-terminal 66His-tag, was constructed as follows. A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO Invitrogen , yielding pWKS1247.", "A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO Invitrogen , yielding pWKS1247. This plasmid was digested with NdeI and XhoI, separated on a 1% agarose/0.56 TAE 20 mM Tris Acetate, 0.5 mM EDTA gel, the fragment corresponding to the DNA binding domain was recovered by gel-isolation using a GeneJET Gel Extraction kit, Fermentas and cloned into similarly digested pMF14 that had been gel-isolated in the same manner. The construct was verified by PCR, restriction analyses and DNA sequencing using primers oWKS-135 and oWKS-136 see below . Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO.", "Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO. The inserts, and in the case of the mutated PabrB promoters the presence of the desired point mutations in the consensus 0A box, were verified by DNA sequencing using primers oWKS-24 and oWKS-25 see below . Sequence grade plasmids were isolated using a Nucleospin Plasmid QuickPure kit Macherey Nagel according to the manufacturer's instructions, except that two lysis reactions were combined onto a single filter and eluted with 65uC prewarmed AE buffer. All constructs were sequenced using BigDye Terminator chemistry Invitrogen on an ABI3130 sequencer Perkin Elmer , according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix Invitrogen in a final volume of 20 mL.", "All constructs were sequenced using BigDye Terminator chemistry Invitrogen on an ABI3130 sequencer Perkin Elmer , according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix Invitrogen in a final volume of 20 mL. After thermocycling, DNA was precipitated and washed with 65% isopropanol, and dissolved in 12 mL HiDi formamid Invitrogen at 96uC for 2 mins and stored in the dark at 4uC until the sequencing run. Sequence analyses were performed in CloneManager Professional Suite 7 SciEd and Geneious version 5.6.2 Biomatters Ltd . Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta DE3 pLysS Novagen . Transformants were used to inoculate 25 mL of LB with appropriate antibiotics.", "Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta DE3 pLysS Novagen . Transformants were used to inoculate 25 mL of LB with appropriate antibiotics. After overnight incubation, the cells were 1:100 diluted in 500 mL fresh medium containing appropriate antibiotics. Protein production was induced with 1 mM IPTG at an OD600 of 0.7 and growth was continued for another three hours before harvesting. Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described .", "Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described . In short, cells were disrupted in 4 mL lysis buffer 2 mM PMSF, 10 mM imidazole, 5 mM beta-mercaptoethanol, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . Cleared cell lysates we incubated with 2 mL pre-equilibrated 50% TALON slurry Clontech in a final volume of 15 mL lysis buffer for 1 hr. The resin was allowed to settle on a Poly-Prep column BioRad and washed with 2 mL wash buffer 20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column identical to wash buffer but with 50, 100, 250 or 500 mM imidazole .", "The resin was allowed to settle on a Poly-Prep column BioRad and washed with 2 mL wash buffer 20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column identical to wash buffer but with 50, 100, 250 or 500 mM imidazole . The whole procedure was carried out at 4uC. Fractions were assayed for purity and yield and suitable fractions were dialysed against 26 1L dialysis buffer 50 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM DTT using Slide-A-Lyzer cassettes with a molecular weight cut-off of 3.5 kDa Pierce . Proteins were stored at 280uC in storage buffer identical to dialysis buffer but containing 20% glycerol . Protein concentrations were determined using Bradford reagent BioRad , according to the manufacturer's instructions.", "Proteins were stored at 280uC in storage buffer identical to dialysis buffer but containing 20% glycerol . Protein concentrations were determined using Bradford reagent BioRad , according to the manufacturer's instructions. DNA fragments for use in EMSA experiment were generated by PCR using GoTaq polymerase Promega and chromosomal DNA from B. subtilis JH642 Bacillus Genetic Stock Center 1A96; plasmids listed in Table 1 , or chromosomal DNA from C. difficile 630Derm as a template. Primers and specific cycling conditions for generation of the EMSA probes are listed in Text S1. DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS and a QIAExII kit Qiagen , according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase Invitrogen according to the instructions of the manufacturer.", "DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS and a QIAExII kit Qiagen , according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase Invitrogen according to the instructions of the manufacturer. Specific activity was determined on a LS6000 scintillation counter Beckman . EMSA conditions were based on previous studies . In short, binding reactions were carried out in binding buffer 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol in the presence of 200 mg/mL bovine serum albumin NEB and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer.", "In short, binding reactions were carried out in binding buffer 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol in the presence of 200 mg/mL bovine serum albumin NEB and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer. Electrophoresis was carried out for 120 min at 85 V. After vacuum drying the gels onto filter paper, they were imaged after overnight exposure on Phosphorimager screens on a Typhoon instrument GE Healthcare . The toxic effects of C. difficile culture supernatants on Vero cells a kind gift of Eric Snijder were determined as follows. Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium Dulbecco modified Eagle medium Lonza supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum , before applying them to a monolayer of Vero cells, and incubation was continued for another hour.", "Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium Dulbecco modified Eagle medium Lonza supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum , before applying them to a monolayer of Vero cells, and incubation was continued for another hour. As a positive control, 50 mL 1:10 diluted purified toxin Techlab was added to the cells. To determine if observed cytotoxic effects were specific for the large clostridial toxins, commercially available anti-toxin against TcdA and TcdB Techlab was added to 10-fold diluted bacterial supernatant for 60 min prior to incubation on the Vero cells. Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects cell rounding were observed. Statistical significance was evaluated with an independent sample t-test.", "Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects cell rounding were observed. Statistical significance was evaluated with an independent sample t-test. Immunization of mice with full length C. difficile Spo0A-6xHis was kindly performed at the Welcome Trust Sanger Institute Hinxton, UK . Cells from 1 mL of C. difficile culture were collected by centrifugation for 1 min at 14000 rpm in a table top centrifuge and resuspended in 200 mL resuspension buffer 10 mM Tris HCl pH 8, 10 mM EDTA, 0.5 mg/mL lysozyme, 1 mM Pefabloc SC Roche . After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer 0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB was added, and samples were heated to 96uC for 5 mins. Total cell lysates amounts corrected for OD 600 were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride PVDF membrane.", "After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer 0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB was added, and samples were heated to 96uC for 5 mins. Total cell lysates amounts corrected for OD 600 were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride PVDF membrane. Membranes were blocked in PBST buffer phosphate buffered saline with 0.1% v/v Tween-20 containing 5% membrane blocking reagent Amersham Biosciences . To visualize Spo0A protein cleared polyclonal serum from a single mouse at a 1:3000 dilution was used, followed by either a goat-anti-mouse HRP-conjugated secondary antibody followed by ECL+ detection Amersham Bioscience , or a goatanti-mouse-biotin-conjugated secondary antibody Dako followed by a tertiary mouse-anti-biotin Cy3-conjugated antibody Jackson . Detection was done using on a Typhoon instrument GE Healthcare . Background corrected peak volumes were quantified using ImageQuant TL Amersham Biosciences .", "Detection was done using on a Typhoon instrument GE Healthcare . Background corrected peak volumes were quantified using ImageQuant TL Amersham Biosciences . Alignments of B. subtilis and C. difficile spo0A were made using ClustalW2 on the basis of the published genome sequences, Genbank accession numbers AL009126 and AM180355, respectively, and the 630Derm spo0A sequence as determined in this study. The sequence for spo0A of C. difficile strain 630Derm was deposited in Genbank accession no JX050222 . Consensus Spo0A boxes were identified using a Single string Search command in Genome2D , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel.", "Consensus Spo0A boxes were identified using a Single string Search command in Genome2D , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel. The results were manually inspected for those boxes within 500 bp upstream of a gene on the same strand. Figures for publication were prepared using ImageQuant TL Amersham Biosciences , Adobe Photoshop CS3 Adobe Systems Inc and Corel Graphics Suite X5 Corel Corporation . In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain DBD were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli Fig. 1A and purified to near homogeneity using metal affinity chromatography Fig.", "In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain DBD were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli Fig. 1A and purified to near homogeneity using metal affinity chromatography Fig. 1A ; lanes P . Full length protein was used to raise antibodies to detect Spo0A in total lysates of C. difficile strains, and the purified DNA binding domain was used in subsequent in vitro binding assays see below . We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells.", "We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells. We performed immunoblotting using polyclonal antibodies against C. difficile Spo0A on total lysates of wild type and spo0A mutant cells grown in a trypton-yeast based medium TTY . We found a clear signal of the size expected for full length Spo0A ,31 kDa as early as 3 hours post inoculation exponential growth phase , through transition phase 8 h as well as 24 and 48 hours post inoculation stationary growth phase Figure 1B; 630Derm . The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant Fig. 1B , CT::spo0A .", "The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant Fig. 1B , CT::spo0A . We obtained similar results in other media, such as the commonly used supplemented brain heart infusion broth BHIS; data not shown . To determine relative levels of Spo0A throughout growth, we performed an immunoblot experiment using fluorescent antibodies, which gives more quantitative information compared to the use of horseradish peroxidase conjugated antibodies in our hands. We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation Figure 1C . Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase.", "We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation Figure 1C . Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase. Spo0A of C. difficile Strain 630Derm Contains a 6aminoacid Duplication BLAST homology searches readily identify a homolog of the well-characterized B. subtilis Spo0A protein in C. difficile 630 CD1214 and previous work demonstrated that a spo0A mutant an insertional inactivation of cd1214 -as expected -no longer forms spores . In silico analyses suggest a similar secondary structure for both proteins Fig. 2A , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region . We compared the sequence of CD1214 obtained from our lab strain 630Derm to that of the published C. difficile 630 genome .", "2A , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region . We compared the sequence of CD1214 obtained from our lab strain 630Derm to that of the published C. difficile 630 genome . Strain 630Derm is a spontaneous erythromycin sensitive strain, which is commonly used in mutagenesis studies and was obtained by serial passaging of strain 630 . The 630Derm spo0A sequence Genbank accession no JX050222 was derived from the expression plasmids constructed for this study, and confirmed in a whole genome sequence of strain 630Derm generated in our lab data not shown . We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid NVGNIE duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation hinge , flanking the highly conserved DNA binding domain Fig.", "We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid NVGNIE duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation hinge , flanking the highly conserved DNA binding domain Fig. 2A and B . We verified the absence of this duplication in strain 630 by PCR Fig. 2C as well as sequencing from the chromosomal DNA of C. difficile 630 data not shown , to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 to which 630 and 630Derm belong by PCR, but the duplication was found to be unique to 630Derm among the isolates tested data not shown .", "2C as well as sequencing from the chromosomal DNA of C. difficile 630 data not shown , to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 to which 630 and 630Derm belong by PCR, but the duplication was found to be unique to 630Derm among the isolates tested data not shown . C. difficile Spo0A-DBD Shows Similar Specificity as B. subtilis Spo0A-DBD Next, we examined the conservation of the DNA binding domain of Spo0A Spo0A-DBD between B. subtilis and C. difficile. In B. subtilis amino acid residues contacting the backbone of the DNA and interacting with specific residues of the Spo0A binding sequence have been defined . We found that all these residues were conserved in the C. difficile protein sequence Fig. 2B , indicating that the protein likely recognizes a similar motif.", "We found that all these residues were conserved in the C. difficile protein sequence Fig. 2B , indicating that the protein likely recognizes a similar motif. DNA binding by full length Spo0A in B. subtilis requires phosphorylation dependent dimerization . However, it was shown that the isolated DBD is capable of binding to legitimate targets of the full length protein . Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene PabrB of B. subtilis.", "Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene PabrB of B. subtilis. PabrB is commonly used as a high-affinity control in binding assays with the B. subtilis Spo0A or Spo0A-DBD protein . It is noteworthy that we failed to identify a homolog of abrB in C. difficile using BLAST, indicating that potential indirect regulation by Spo0A cannot occur through abrB in C. difficile as it does in B. subtilis. We found that C. difficile Spo0A-DBD bound with high affinity to PabrB Fig. 2D and E .", "We found that C. difficile Spo0A-DBD bound with high affinity to PabrB Fig. 2D and E . We performed electrophoretic mobility shift assays EMSAs using radiolabeled PabrB and increasing amounts of purified C. difficile Spo0A-DBD that was purified using a C-terminal 66His-tag. The addition of protein leads to a dose-dependent retardation of the DNA fragment with an apparent K D of ,50 nM. In the same range of protein concentrations, no binding was observed for a negative control a DNA fragment of B. subtilis citG Fig. 2E , suggesting that binding was specific for the abrB promoter region. B. subtilis Spo0A recognizes a distinct sequence 0A box , that is characterized by a 7 bp core motif TGTCGAA .", "2E , suggesting that binding was specific for the abrB promoter region. B. subtilis Spo0A recognizes a distinct sequence 0A box , that is characterized by a 7 bp core motif TGTCGAA . Structural studies have revealed that the protein makes specific contacts with the G at position 2 G2 , and the C at position 4 C4 and 5 G5 of this motif . We introduced G2A, C4A, G5A, G2A/C4A and C4A/G5A mutations in the perfect consensus core 0A-box present in PabrB. We found that the affinity of C. difficile Spo0A for these mutated PabrB fragments was highly reduced Fig. 2E . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence.", "2E . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence. For the single point mutations in the DNA, the affinity decreased ,10-fold. There did not seem to be an additive effect of a second point mutation for the two combinations tested. None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other non-consensus 0A boxes in the abrB promoter . Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD.", "None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other non-consensus 0A boxes in the abrB promoter . Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD. Value for Binding by C. difficile Spo0A-DBD Above, we have established that the Spo0A-DBD of C. difficile is highly homologous to that of the B. subtilis Spo0A protein, and that the proteins recognize a similar consensus sequence Fig. 2 . Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A. We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D .", "Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A. We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D . Such an analysis revealed the presence of 102 matching motifs, of which 45 were located within 500 bp of the initiating ATG of an open reading frame on the same strand see Table S1 . Our attention was drawn to spo0A and sigH, as these two genes were previously found to be regulated by Spo0A in B. subtilis and/or play important roles in sporulation 3, . We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH. We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames.", "We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH. We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames. We found that the addition of Spo0A-DBD to the reactions caused retardation of the spo0A and sigH DNA fragments Fig. 3A , but not of a spoVG fragment which did not contain a consensus 0A box Fig. 3B . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA.", "3B . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA. Moreover, the presence of multiple shifted species could indicate the presence of more than one strong binding site. These results establish that spo0A and sigH are likely legitimate targets of Spo0A in C. difficile, and confirm that spoVG is not, in line with results obtained in B. subtilis . We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon.", "We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon. This positioning is similar to that observed for spo0A . and sigH .. We confirmed in vitro binding of the C. difficile Spo0A-DBD to the promoter regions of lplA and ssuA. We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene CD1654; box at 267 and the ssuA gene CD1484; box at 282 Fig.", "We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene CD1654; box at 267 and the ssuA gene CD1484; box at 282 Fig. 3A . The lplA gene encodes a predicted lipoate-protein ligase, and ssuA is annotated as an aliphatic sulfonates ABC transporter; to our knowledge, neither of these have been directly implicated in sporulation or have found to be targets for Spo0A in other organisms. Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile. It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway .", "Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile. It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway . However, the in silico identification of upstream regions with a consensus Spo0A binding site did not point to any of the early sporulation genes downstream of spo0A itself as direct targets of Spo0A. This is likely the result of variations in the 0A-box in these promoters that were disregarded in the box search. In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A such as spoIIAA and spoIIE do not contain a 100% match to the core motif, but rather one or more near-consensus boxes . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above Fig.", "In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A such as spoIIAA and spoIIE do not contain a 100% match to the core motif, but rather one or more near-consensus boxes . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above Fig. 3B . For spoIIAA encoding an antianti sigma-factor and spoIIE encoding a serine phosphatase , we observed a low intensity shifted species at concentrations as low as 150 nM. For spoIIGA encoding a sporulation specific protease we observed the shifted species only at higher concentrations of protein .200 nM . The negative control spoVG did not demonstrate binding of Spo0A-DBD at these concentrations.", "For spoIIGA encoding a sporulation specific protease we observed the shifted species only at higher concentrations of protein .200 nM . The negative control spoVG did not demonstrate binding of Spo0A-DBD at these concentrations. Moreover, the shift we observed was reversible using unlabeled DNA containing a high affinity binding site, but not using unlabeled DNA that lacked such a site Figure S1B-D . Therefore, we consider the binding to spoIIAA, spoIIE and spoIIGA genes to be specific, despite the fact that increasing the amount of protein did not seem to cause a significant increase in the amount of DNA in the complex. Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions. C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells .", "Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions. C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells . Considering the absence of a homolog of the abrB repressor, direct binding of Spo0A and concomitant activation of toxin gene transcription is a likely mechanism through which this could occur. We found evidence for direct binding of Spo0A-DBD to the region upstream of tcdB, encoding one of the major clostridial toxin genes, and possibly tcdC, but this did not seem to result in lower toxin levels in our hands. We performed EMSAs using DNA upstream of tcdR encoding a sigma factor responsible for the activation of toxin gene transcription , tcdB encoding toxin B , tcdA encoding toxin A . In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE encoding a holin-like protein and tcdC encoding a putative negative regulator of toxin production , even though this regulator does not have a significant effect on toxin levels under the conditions we used .", "We performed EMSAs using DNA upstream of tcdR encoding a sigma factor responsible for the activation of toxin gene transcription , tcdB encoding toxin B , tcdA encoding toxin A . In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE encoding a holin-like protein and tcdC encoding a putative negative regulator of toxin production , even though this regulator does not have a significant effect on toxin levels under the conditions we used . Of the regions tested, we only observed a clear shifted species, indicative of Spo0A binding, for tcdB Figure 4A ; the shifted species in our EMSA assay was reversed by the addition of unlabeled DNA containing a high affinity binding site, but not by DNA lacking such a site Figure S1E . For tcdC, some smearing was observed at all concentrations of proteins tested Figure 4A , and there did not seem to be a clear effect of the addition of unlabeled DNA fragments Figure S1F . The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG. We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested.", "The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG. We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested. We found no lower toxicity towards Vero cells of culture supernatants derived from spo0A mutant cells compared to wild type. We grew three independent biological replicates of a wild type 630Derm or Clostron-generated spo0A mutant CT::spo0A -a kind gift of the Minton lab in glucose-free TTY medium. We harvested culture supernatant at late-exponential phase approximately 7 hours post inoculation , the transition phase between exponential and stationary growth phase approximately 9 hours post inoculation , as well as two time points in stationary phase 24 and 48 hours post inoculation and determined the toxin endpoint titres see Materials and Methods . In contrast to previous findings, we observed a small #4-fold increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant p.0.05, independent sample t-test .", "We harvested culture supernatant at late-exponential phase approximately 7 hours post inoculation , the transition phase between exponential and stationary growth phase approximately 9 hours post inoculation , as well as two time points in stationary phase 24 and 48 hours post inoculation and determined the toxin endpoint titres see Materials and Methods . In contrast to previous findings, we observed a small #4-fold increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant p.0.05, independent sample t-test . In other medium BHIS , we observed no differences at all data not shown . We conclude that Spo0A does not positively affect toxin production in C. difficile 630Derm and the in vivo relevance of the binding to regions upstream of tcdB and/or tcdC is therefore limited under our experimental conditions. The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box .", "The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box . Depending on the analysis, this motif is flanked by one or more adenine or thymine residues . Interestingly, many target genes do not harbor a perfect match to this consensus sequence, but rather contain one or more degenerate motifs. The differences in these motifs may reflect different promoter architectures e.g. AT content , modes of action e.g.", "The differences in these motifs may reflect different promoter architectures e.g. AT content , modes of action e.g. activation or repression or levels of regulation. Spo0A genes in B. subtilis can be divided in different classes that respond to different levels of phosphorylated Spo0A . For C. difficile, we conclude that the Spo0A protein likely recognizes a motif that is similar to the B. subtilis Spo0A box on the basis of four lines of evidence; 1. All DNA binding/contacting residues are conserved Fig. 2B , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A Fig. 2D , 3.", "2B , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A Fig. 2D , 3. Mutagenesis of key residues in the B. subtilis Spo0A box reduces affinity of C. difficile Spo0A for DNA Fig. 2E and 4. A B. subtilis Spo0A box has predictive value for DNA binding by C. difficile Spo0A Fig. 3A . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein.", "3A . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein. It is likely that differences do exist between the preferred binding sites for both proteins that will be evident when a comprehensive analysis is performed of in vivo DNA binding of C. difficile Spo0A; based on the limited data set of this study, a MEME analysis already suggests possible differences in the extended Spo0A motif W.K. Smits, unpublished observations . These differences may relate to the much higher AT content of C. difficile compared to B. subtilis 71 vs. 56.5%, respectively , or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation for review see ref .", "These differences may relate to the much higher AT content of C. difficile compared to B. subtilis 71 vs. 56.5%, respectively , or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation for review see ref . The activation of Spo0A is controlled by a multi-component phosphorelay that can integrate environmental cues and ensures a gradual increase in the level of phosphorylated Spo0A in the cell . In addition, the transcription of the spo0A gene is controlled by multiple feedback loops. For instance, Spo0A regulates its own transcription by binding to the spo0A promoter , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter . In C. difficile, there are some interesting differences and similarities in the regulatory pathways.", "For instance, Spo0A regulates its own transcription by binding to the spo0A promoter , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter . In C. difficile, there are some interesting differences and similarities in the regulatory pathways. Most notably, there seems to be no phosphorelay and the phosphorylation state of Spo0A is supposedly controlled by orphan histidine kinases . The transcription of spo0A in C. difficile is under control of the transition state sigma factor Sigma H , as it is in B. subtilis . Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile Fig. 3A , raising the possibility of auto-regulation of spo0A.", "Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile Fig. 3A , raising the possibility of auto-regulation of spo0A. The putative direct regulation of sigH by Spo0A may reflect that the C. difficile genome does not harbor a homolog of the pleiotropic regulator AbrB, which is responsible for the Spo0A-dependent regulation of sigH in B. subtilis . Consistent with a model in which spo0A is positively autoregulated, we noted a sharp increase in the levels of Spo0A as cells approach the stationary growth phase Figure 1C . Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA Fig. 3B .", "Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA Fig. 3B . All these observations are consistent with direct regulation of these genes by Spo0A in other organisms , and the conservation of the sporulation pathway . Though Spo0A is the key regulator for sporulation in Firmicutes, it regulates numerous other processes in various bacteria. In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology 10, . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB.", "In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology 10, . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB. Additionally, transcription of abrB responds already to low levels of Spo0A,P . As a result these effects are detectable in late-exponential and early stationary phase, as some Spo0A is present throughout growth in B. subtilis cells. Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect.", "Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect. In Clostridium acetobutylicum and C. beijerinckii, Spo0A is a direct regulator of solvent formation, as well as sporulation . It seems therefore conceivable that Spo0A in C. difficile also affects aspects of metabolism. In this respect, it is important to note that also in C. difficile Spo0A is detectable from early exponential growth phase on Figure 1B . We observed direct binding of C. difficile Spo0A to the promoter region of sigH Fig. 3A .", "We observed direct binding of C. difficile Spo0A to the promoter region of sigH Fig. 3A . This gene encodes the key sigma factor for the transition phase, and regulates processes outside sporulation as well . Moreover, we found significant levels of Spo0A from early stationary phase on Fig. 1B and unpublished observations , indicating the regulatory actions of Spo0A need not be limited to stationary phase in C. difficile. In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA Fig. 3A .", "In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA Fig. 3A . The presence of a putative Spo0A binding site upstream of these genes, as well as the spacing compared to the start codon, is conserved in the problematic Stoke-Mandeville strain R20291 , a member of PCR ribotype 27. This could indicate that these aspects of regulation by Spo0A are conserved in multiple strains of C. difficile. It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed.", "It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed. In order to distinguish direct from indirect effects, in vivo binding profiles of Spo0A should be performed. The antibodies generated for this study should prove to be useful for this type of experiments. Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control .", "Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control . Similarly, the production of the emetic toxin cereulide in B. cereus is greatly repressed in a spo0A mutant, in an AbrB-dependent manner . In contrast, Spo0A directly represses the expression of the cry toxin genes in B. thuringiensis and a spo0A mutant is therefore a hyper-producer of the insecticidal crystal protein . In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A and also the production of enterotoxin in this organism seems to be indirectly dependent on sporulation . In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A TcdA , both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B TcdB .", "In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A and also the production of enterotoxin in this organism seems to be indirectly dependent on sporulation . In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A TcdA , both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B TcdB . Our in vitro binding data indicate a potential binding site for Spo0A upstream of tcdB and possibly tcdC Fig. 4A . However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant a kind gift of the Minton lab; did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type #2fold in exponential phase cells up to 4-fold in late-stationary phase cells .", "However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant a kind gift of the Minton lab; did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type #2fold in exponential phase cells up to 4-fold in late-stationary phase cells . The small, and not significant, differences in toxin levels in our experiments might be attributed to differences in the susceptibility of cells for lysis rather than the production of toxin, but could also indicate a negative regulatory effect of Spo0A on toxin production. In support of the latter hypothesis, it was recently reported that a spo0A mutant of C. difficile strain R20291 a PCR ribotypes 027/BI/NAP1 epidemic strain demonstrates ,10fold higher toxin levels than its isogenic wild type 30 h post inoculation, and is significantly more virulent in a mouse model of disease . The differences between Underwood et al on the one hand and our study as well as the study of Deakin and coworkers on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously data not shown .", "The differences between Underwood et al on the one hand and our study as well as the study of Deakin and coworkers on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously data not shown . Alternatively, the differences could indicate integration of the group II intron at more than one location in the chromosome in the strain used in Underwood et al . In the absence of a complementation experiment and/or Southern blot data, this remains to be established. In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays.", "In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays. In the present study we have for the first time demonstrated direct binding of the DNA binding domain of C. difficile Spo0A to putative target DNA. This work has revealed that aspects of Spo0A binding are conserved between Bacillus and C. difficile 0A box, possible auto-regulation and binding to early sporulation promoters , whereas others are not the absence of abrB as a direct target in C. difficile, binding to DNA upstream of lplA, ssuA . The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo.", "The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo. The identification of genes affected by Spo0A in C. difficile may shed light on the role of the protein in virulence and pathogenesis of this organism. Figure S1 Specificity controls for binding by Spo0A-DBD-his6. Arrows indicate the position of shifted species DNA:protein complexes . Titrations with PCR fragments of PabrB containing a high affinity binding site and PtcdA lacking such a site correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A.", "Titrations with PCR fragments of PabrB containing a high affinity binding site and PtcdA lacking such a site correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A. Comparison of binding of Spo0A-DBD-his6, Spo0A-his6 and CD2195-his6 binding to the upstream region of spoIIAA. B. Binding of Spo0A-DBD-his6 to the upstream region of spoIIAA is reversed by the addition of PabrB, but not by the addition of PtcdA . C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA.", "C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA. E. Binding of Spo0A-DBD-his6 to the upstream region of tcdB is reversed by the addition of PabrB, but not by the addition of PtcdA. F. Binding of Spo0A-DBD-his6 to the upstream region of tcdC is not or moderately affected by the addition of PabrB and/or PtcdA. TIF Text S1 Oligonucleotides used in this study and PCR cycling conditions for the EMSA probes. PDF" ]

[ "Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection CDI , for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630.", "Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct putative targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds.", "These results identify for the first time direct putative targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds. Spo0A is the key regulator for sporulation . Most of our knowledge about the protein is based on work in Bacilli. Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay .", "Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay . The signaling cascade allows for the integration of environmental signals into the regulation of Spo0A dependent processes, including sporulation. The two functional domains, the N-terminal phosphorylation and dimerization domain receiver domain , and the C-terminal DNA binding effector domain are separated by a hinge region that is relatively poorly conserved . Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation .", "Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation . Extensive characterization of Spo0A targets has revealed a motif that represents a high affinity Spo0A binding site, the 0A box . The crystal structure of the DNA binding domain confirms specific and non-specific contacts between the protein and the consensus sequence . It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis , and solvent production in Clostridium acetobutylicum . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection CDI for recent reviews see .", "It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis , and solvent production in Clostridium acetobutylicum . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection CDI for recent reviews see . Though many people are asymptomatically colonized by C. difficile, the bacterium can cause serious health problems, such as pseudomembranous colitis and toxic megacolon, under the influence of risk factors such as age and antibiotic use. As a result, CDI was long regarded a nosocomial infection. Recently, however, an increase in the cases of community acquired CDI can be observed . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 BI/ NAP1 and 078 .", "Recently, however, an increase in the cases of community acquired CDI can be observed . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 BI/ NAP1 and 078 . Its main virulence factors are the major clostridial toxins A and B . In addition, certain strains of C. difficile, including ribotypes 027 and 078, additionally encode a binary toxin . C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow .", "C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow . There is limited knowledge about the regulation of sporulation in C. difficile. It has been reported that spo0A, as expected, is required for the formation of spores and the gene is required for persistence and transmission in mice . Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A .", "Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A . Similarly, it was reported that multiple orphan histidine kinases can phosphorylate Spo0A in C. acetobutylicum . Recently, it was reported that spo0A can be transcribed from a SigH-dependent promoter . It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes.", "It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes. Escherichia coli strains were routinely grown in Luria-Bertani broth or plates, supplemented with appropriate antibiotics. Chloramphenicol was used at a final concentration of 20 mg/mL for agar plates and 10 mg/mL for liquid cultures. Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL.", "Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL. Cloning was carried out using E. coli DH5a, overexpression was performed in E. coli Rosetta DE3 pLysS Novagen . C. difficile strains were grown in a glucose-free trypton-yeast based medium TTY; 3% w/v bactotrypton BD , 2% yeast extract Fluka , 0.1% w/v thioglycollate Sigma pH 7.4 , supplemented with 20 mg/mL of lincomycin when appropriate, or on CLO or TSS plates Biomerieux . All plasmids are listed in Table 1 . Primers obtained from Sigma Aldrich are listed in Text S1 and specific cycling conditions are available on request.", "All plasmids are listed in Table 1 . Primers obtained from Sigma Aldrich are listed in Text S1 and specific cycling conditions are available on request. Unless noted otherwise, PCR reactions were carried out using Pfu polymerase Fermentas according to the instructions of the manufacturer. Plasmid pWKS1251, for the overproduction of Spo0A-DBD carrying a C-terminal 66His-tag, was constructed as follows. A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO Invitrogen , yielding pWKS1247.", "A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO Invitrogen , yielding pWKS1247. This plasmid was digested with NdeI and XhoI, separated on a 1% agarose/0.56 TAE 20 mM Tris Acetate, 0.5 mM EDTA gel, the fragment corresponding to the DNA binding domain was recovered by gel-isolation using a GeneJET Gel Extraction kit, Fermentas and cloned into similarly digested pMF14 that had been gel-isolated in the same manner. The construct was verified by PCR, restriction analyses and DNA sequencing using primers oWKS-135 and oWKS-136 see below . Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO.", "Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO. The inserts, and in the case of the mutated PabrB promoters the presence of the desired point mutations in the consensus 0A box, were verified by DNA sequencing using primers oWKS-24 and oWKS-25 see below . Sequence grade plasmids were isolated using a Nucleospin Plasmid QuickPure kit Macherey Nagel according to the manufacturer's instructions, except that two lysis reactions were combined onto a single filter and eluted with 65uC prewarmed AE buffer. All constructs were sequenced using BigDye Terminator chemistry Invitrogen on an ABI3130 sequencer Perkin Elmer , according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix Invitrogen in a final volume of 20 mL.", "All constructs were sequenced using BigDye Terminator chemistry Invitrogen on an ABI3130 sequencer Perkin Elmer , according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix Invitrogen in a final volume of 20 mL. After thermocycling, DNA was precipitated and washed with 65% isopropanol, and dissolved in 12 mL HiDi formamid Invitrogen at 96uC for 2 mins and stored in the dark at 4uC until the sequencing run. Sequence analyses were performed in CloneManager Professional Suite 7 SciEd and Geneious version 5.6.2 Biomatters Ltd . Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta DE3 pLysS Novagen . Transformants were used to inoculate 25 mL of LB with appropriate antibiotics.", "Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta DE3 pLysS Novagen . Transformants were used to inoculate 25 mL of LB with appropriate antibiotics. After overnight incubation, the cells were 1:100 diluted in 500 mL fresh medium containing appropriate antibiotics. Protein production was induced with 1 mM IPTG at an OD600 of 0.7 and growth was continued for another three hours before harvesting. Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described .", "Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described . In short, cells were disrupted in 4 mL lysis buffer 2 mM PMSF, 10 mM imidazole, 5 mM beta-mercaptoethanol, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . Cleared cell lysates we incubated with 2 mL pre-equilibrated 50% TALON slurry Clontech in a final volume of 15 mL lysis buffer for 1 hr. The resin was allowed to settle on a Poly-Prep column BioRad and washed with 2 mL wash buffer 20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column identical to wash buffer but with 50, 100, 250 or 500 mM imidazole .", "The resin was allowed to settle on a Poly-Prep column BioRad and washed with 2 mL wash buffer 20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column identical to wash buffer but with 50, 100, 250 or 500 mM imidazole . The whole procedure was carried out at 4uC. Fractions were assayed for purity and yield and suitable fractions were dialysed against 26 1L dialysis buffer 50 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM DTT using Slide-A-Lyzer cassettes with a molecular weight cut-off of 3.5 kDa Pierce . Proteins were stored at 280uC in storage buffer identical to dialysis buffer but containing 20% glycerol . Protein concentrations were determined using Bradford reagent BioRad , according to the manufacturer's instructions.", "Proteins were stored at 280uC in storage buffer identical to dialysis buffer but containing 20% glycerol . Protein concentrations were determined using Bradford reagent BioRad , according to the manufacturer's instructions. DNA fragments for use in EMSA experiment were generated by PCR using GoTaq polymerase Promega and chromosomal DNA from B. subtilis JH642 Bacillus Genetic Stock Center 1A96; plasmids listed in Table 1 , or chromosomal DNA from C. difficile 630Derm as a template. Primers and specific cycling conditions for generation of the EMSA probes are listed in Text S1. DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS and a QIAExII kit Qiagen , according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase Invitrogen according to the instructions of the manufacturer.", "DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS and a QIAExII kit Qiagen , according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase Invitrogen according to the instructions of the manufacturer. Specific activity was determined on a LS6000 scintillation counter Beckman . EMSA conditions were based on previous studies . In short, binding reactions were carried out in binding buffer 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol in the presence of 200 mg/mL bovine serum albumin NEB and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer.", "In short, binding reactions were carried out in binding buffer 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol in the presence of 200 mg/mL bovine serum albumin NEB and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer. Electrophoresis was carried out for 120 min at 85 V. After vacuum drying the gels onto filter paper, they were imaged after overnight exposure on Phosphorimager screens on a Typhoon instrument GE Healthcare . The toxic effects of C. difficile culture supernatants on Vero cells a kind gift of Eric Snijder were determined as follows. Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium Dulbecco modified Eagle medium Lonza supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum , before applying them to a monolayer of Vero cells, and incubation was continued for another hour.", "Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium Dulbecco modified Eagle medium Lonza supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum , before applying them to a monolayer of Vero cells, and incubation was continued for another hour. As a positive control, 50 mL 1:10 diluted purified toxin Techlab was added to the cells. To determine if observed cytotoxic effects were specific for the large clostridial toxins, commercially available anti-toxin against TcdA and TcdB Techlab was added to 10-fold diluted bacterial supernatant for 60 min prior to incubation on the Vero cells. Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects cell rounding were observed. Statistical significance was evaluated with an independent sample t-test.", "Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects cell rounding were observed. Statistical significance was evaluated with an independent sample t-test. Immunization of mice with full length C. difficile Spo0A-6xHis was kindly performed at the Welcome Trust Sanger Institute Hinxton, UK . Cells from 1 mL of C. difficile culture were collected by centrifugation for 1 min at 14000 rpm in a table top centrifuge and resuspended in 200 mL resuspension buffer 10 mM Tris HCl pH 8, 10 mM EDTA, 0.5 mg/mL lysozyme, 1 mM Pefabloc SC Roche . After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer 0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB was added, and samples were heated to 96uC for 5 mins. Total cell lysates amounts corrected for OD 600 were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride PVDF membrane.", "After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer 0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB was added, and samples were heated to 96uC for 5 mins. Total cell lysates amounts corrected for OD 600 were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride PVDF membrane. Membranes were blocked in PBST buffer phosphate buffered saline with 0.1% v/v Tween-20 containing 5% membrane blocking reagent Amersham Biosciences . To visualize Spo0A protein cleared polyclonal serum from a single mouse at a 1:3000 dilution was used, followed by either a goat-anti-mouse HRP-conjugated secondary antibody followed by ECL+ detection Amersham Bioscience , or a goatanti-mouse-biotin-conjugated secondary antibody Dako followed by a tertiary mouse-anti-biotin Cy3-conjugated antibody Jackson . Detection was done using on a Typhoon instrument GE Healthcare . Background corrected peak volumes were quantified using ImageQuant TL Amersham Biosciences .", "Detection was done using on a Typhoon instrument GE Healthcare . Background corrected peak volumes were quantified using ImageQuant TL Amersham Biosciences . Alignments of B. subtilis and C. difficile spo0A were made using ClustalW2 on the basis of the published genome sequences, Genbank accession numbers AL009126 and AM180355, respectively, and the 630Derm spo0A sequence as determined in this study. The sequence for spo0A of C. difficile strain 630Derm was deposited in Genbank accession no JX050222 . Consensus Spo0A boxes were identified using a Single string Search command in Genome2D , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel.", "Consensus Spo0A boxes were identified using a Single string Search command in Genome2D , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel. The results were manually inspected for those boxes within 500 bp upstream of a gene on the same strand. Figures for publication were prepared using ImageQuant TL Amersham Biosciences , Adobe Photoshop CS3 Adobe Systems Inc and Corel Graphics Suite X5 Corel Corporation . In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain DBD were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli Fig. 1A and purified to near homogeneity using metal affinity chromatography Fig.", "In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain DBD were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli Fig. 1A and purified to near homogeneity using metal affinity chromatography Fig. 1A ; lanes P . Full length protein was used to raise antibodies to detect Spo0A in total lysates of C. difficile strains, and the purified DNA binding domain was used in subsequent in vitro binding assays see below . We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells.", "We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells. We performed immunoblotting using polyclonal antibodies against C. difficile Spo0A on total lysates of wild type and spo0A mutant cells grown in a trypton-yeast based medium TTY . We found a clear signal of the size expected for full length Spo0A ,31 kDa as early as 3 hours post inoculation exponential growth phase , through transition phase 8 h as well as 24 and 48 hours post inoculation stationary growth phase Figure 1B; 630Derm . The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant Fig. 1B , CT::spo0A .", "The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant Fig. 1B , CT::spo0A . We obtained similar results in other media, such as the commonly used supplemented brain heart infusion broth BHIS; data not shown . To determine relative levels of Spo0A throughout growth, we performed an immunoblot experiment using fluorescent antibodies, which gives more quantitative information compared to the use of horseradish peroxidase conjugated antibodies in our hands. We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation Figure 1C . Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase.", "We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation Figure 1C . Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase. Spo0A of C. difficile Strain 630Derm Contains a 6aminoacid Duplication BLAST homology searches readily identify a homolog of the well-characterized B. subtilis Spo0A protein in C. difficile 630 CD1214 and previous work demonstrated that a spo0A mutant an insertional inactivation of cd1214 -as expected -no longer forms spores . In silico analyses suggest a similar secondary structure for both proteins Fig. 2A , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region . We compared the sequence of CD1214 obtained from our lab strain 630Derm to that of the published C. difficile 630 genome .", "2A , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region . We compared the sequence of CD1214 obtained from our lab strain 630Derm to that of the published C. difficile 630 genome . Strain 630Derm is a spontaneous erythromycin sensitive strain, which is commonly used in mutagenesis studies and was obtained by serial passaging of strain 630 . The 630Derm spo0A sequence Genbank accession no JX050222 was derived from the expression plasmids constructed for this study, and confirmed in a whole genome sequence of strain 630Derm generated in our lab data not shown . We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid NVGNIE duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation hinge , flanking the highly conserved DNA binding domain Fig.", "We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid NVGNIE duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation hinge , flanking the highly conserved DNA binding domain Fig. 2A and B . We verified the absence of this duplication in strain 630 by PCR Fig. 2C as well as sequencing from the chromosomal DNA of C. difficile 630 data not shown , to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 to which 630 and 630Derm belong by PCR, but the duplication was found to be unique to 630Derm among the isolates tested data not shown .", "2C as well as sequencing from the chromosomal DNA of C. difficile 630 data not shown , to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 to which 630 and 630Derm belong by PCR, but the duplication was found to be unique to 630Derm among the isolates tested data not shown . C. difficile Spo0A-DBD Shows Similar Specificity as B. subtilis Spo0A-DBD Next, we examined the conservation of the DNA binding domain of Spo0A Spo0A-DBD between B. subtilis and C. difficile. In B. subtilis amino acid residues contacting the backbone of the DNA and interacting with specific residues of the Spo0A binding sequence have been defined . We found that all these residues were conserved in the C. difficile protein sequence Fig. 2B , indicating that the protein likely recognizes a similar motif.", "We found that all these residues were conserved in the C. difficile protein sequence Fig. 2B , indicating that the protein likely recognizes a similar motif. DNA binding by full length Spo0A in B. subtilis requires phosphorylation dependent dimerization . However, it was shown that the isolated DBD is capable of binding to legitimate targets of the full length protein . Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene PabrB of B. subtilis.", "Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene PabrB of B. subtilis. PabrB is commonly used as a high-affinity control in binding assays with the B. subtilis Spo0A or Spo0A-DBD protein . It is noteworthy that we failed to identify a homolog of abrB in C. difficile using BLAST, indicating that potential indirect regulation by Spo0A cannot occur through abrB in C. difficile as it does in B. subtilis. We found that C. difficile Spo0A-DBD bound with high affinity to PabrB Fig. 2D and E .", "We found that C. difficile Spo0A-DBD bound with high affinity to PabrB Fig. 2D and E . We performed electrophoretic mobility shift assays EMSAs using radiolabeled PabrB and increasing amounts of purified C. difficile Spo0A-DBD that was purified using a C-terminal 66His-tag. The addition of protein leads to a dose-dependent retardation of the DNA fragment with an apparent K D of ,50 nM. In the same range of protein concentrations, no binding was observed for a negative control a DNA fragment of B. subtilis citG Fig. 2E , suggesting that binding was specific for the abrB promoter region. B. subtilis Spo0A recognizes a distinct sequence 0A box , that is characterized by a 7 bp core motif TGTCGAA .", "2E , suggesting that binding was specific for the abrB promoter region. B. subtilis Spo0A recognizes a distinct sequence 0A box , that is characterized by a 7 bp core motif TGTCGAA . Structural studies have revealed that the protein makes specific contacts with the G at position 2 G2 , and the C at position 4 C4 and 5 G5 of this motif . We introduced G2A, C4A, G5A, G2A/C4A and C4A/G5A mutations in the perfect consensus core 0A-box present in PabrB. We found that the affinity of C. difficile Spo0A for these mutated PabrB fragments was highly reduced Fig. 2E . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence.", "2E . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence. For the single point mutations in the DNA, the affinity decreased ,10-fold. There did not seem to be an additive effect of a second point mutation for the two combinations tested. None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other non-consensus 0A boxes in the abrB promoter . Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD.", "None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other non-consensus 0A boxes in the abrB promoter . Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD. Value for Binding by C. difficile Spo0A-DBD Above, we have established that the Spo0A-DBD of C. difficile is highly homologous to that of the B. subtilis Spo0A protein, and that the proteins recognize a similar consensus sequence Fig. 2 . Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A. We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D .", "Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A. We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D . Such an analysis revealed the presence of 102 matching motifs, of which 45 were located within 500 bp of the initiating ATG of an open reading frame on the same strand see Table S1 . Our attention was drawn to spo0A and sigH, as these two genes were previously found to be regulated by Spo0A in B. subtilis and/or play important roles in sporulation 3, . We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH. We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames.", "We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH. We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames. We found that the addition of Spo0A-DBD to the reactions caused retardation of the spo0A and sigH DNA fragments Fig. 3A , but not of a spoVG fragment which did not contain a consensus 0A box Fig. 3B . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA.", "3B . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA. Moreover, the presence of multiple shifted species could indicate the presence of more than one strong binding site. These results establish that spo0A and sigH are likely legitimate targets of Spo0A in C. difficile, and confirm that spoVG is not, in line with results obtained in B. subtilis . We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon.", "We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon. This positioning is similar to that observed for spo0A . and sigH .. We confirmed in vitro binding of the C. difficile Spo0A-DBD to the promoter regions of lplA and ssuA. We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene CD1654; box at 267 and the ssuA gene CD1484; box at 282 Fig.", "We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene CD1654; box at 267 and the ssuA gene CD1484; box at 282 Fig. 3A . The lplA gene encodes a predicted lipoate-protein ligase, and ssuA is annotated as an aliphatic sulfonates ABC transporter; to our knowledge, neither of these have been directly implicated in sporulation or have found to be targets for Spo0A in other organisms. Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile. It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway .", "Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile. It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway . However, the in silico identification of upstream regions with a consensus Spo0A binding site did not point to any of the early sporulation genes downstream of spo0A itself as direct targets of Spo0A. This is likely the result of variations in the 0A-box in these promoters that were disregarded in the box search. In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A such as spoIIAA and spoIIE do not contain a 100% match to the core motif, but rather one or more near-consensus boxes . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above Fig.", "In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A such as spoIIAA and spoIIE do not contain a 100% match to the core motif, but rather one or more near-consensus boxes . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above Fig. 3B . For spoIIAA encoding an antianti sigma-factor and spoIIE encoding a serine phosphatase , we observed a low intensity shifted species at concentrations as low as 150 nM. For spoIIGA encoding a sporulation specific protease we observed the shifted species only at higher concentrations of protein .200 nM . The negative control spoVG did not demonstrate binding of Spo0A-DBD at these concentrations.", "For spoIIGA encoding a sporulation specific protease we observed the shifted species only at higher concentrations of protein .200 nM . The negative control spoVG did not demonstrate binding of Spo0A-DBD at these concentrations. Moreover, the shift we observed was reversible using unlabeled DNA containing a high affinity binding site, but not using unlabeled DNA that lacked such a site Figure S1B-D . Therefore, we consider the binding to spoIIAA, spoIIE and spoIIGA genes to be specific, despite the fact that increasing the amount of protein did not seem to cause a significant increase in the amount of DNA in the complex. Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions. C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells .", "Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions. C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells . Considering the absence of a homolog of the abrB repressor, direct binding of Spo0A and concomitant activation of toxin gene transcription is a likely mechanism through which this could occur. We found evidence for direct binding of Spo0A-DBD to the region upstream of tcdB, encoding one of the major clostridial toxin genes, and possibly tcdC, but this did not seem to result in lower toxin levels in our hands. We performed EMSAs using DNA upstream of tcdR encoding a sigma factor responsible for the activation of toxin gene transcription , tcdB encoding toxin B , tcdA encoding toxin A . In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE encoding a holin-like protein and tcdC encoding a putative negative regulator of toxin production , even though this regulator does not have a significant effect on toxin levels under the conditions we used .", "We performed EMSAs using DNA upstream of tcdR encoding a sigma factor responsible for the activation of toxin gene transcription , tcdB encoding toxin B , tcdA encoding toxin A . In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE encoding a holin-like protein and tcdC encoding a putative negative regulator of toxin production , even though this regulator does not have a significant effect on toxin levels under the conditions we used . Of the regions tested, we only observed a clear shifted species, indicative of Spo0A binding, for tcdB Figure 4A ; the shifted species in our EMSA assay was reversed by the addition of unlabeled DNA containing a high affinity binding site, but not by DNA lacking such a site Figure S1E . For tcdC, some smearing was observed at all concentrations of proteins tested Figure 4A , and there did not seem to be a clear effect of the addition of unlabeled DNA fragments Figure S1F . The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG. We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested.", "The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG. We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested. We found no lower toxicity towards Vero cells of culture supernatants derived from spo0A mutant cells compared to wild type. We grew three independent biological replicates of a wild type 630Derm or Clostron-generated spo0A mutant CT::spo0A -a kind gift of the Minton lab in glucose-free TTY medium. We harvested culture supernatant at late-exponential phase approximately 7 hours post inoculation , the transition phase between exponential and stationary growth phase approximately 9 hours post inoculation , as well as two time points in stationary phase 24 and 48 hours post inoculation and determined the toxin endpoint titres see Materials and Methods . In contrast to previous findings, we observed a small #4-fold increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant p.0.05, independent sample t-test .", "We harvested culture supernatant at late-exponential phase approximately 7 hours post inoculation , the transition phase between exponential and stationary growth phase approximately 9 hours post inoculation , as well as two time points in stationary phase 24 and 48 hours post inoculation and determined the toxin endpoint titres see Materials and Methods . In contrast to previous findings, we observed a small #4-fold increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant p.0.05, independent sample t-test . In other medium BHIS , we observed no differences at all data not shown . We conclude that Spo0A does not positively affect toxin production in C. difficile 630Derm and the in vivo relevance of the binding to regions upstream of tcdB and/or tcdC is therefore limited under our experimental conditions. The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box .", "The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box . Depending on the analysis, this motif is flanked by one or more adenine or thymine residues . Interestingly, many target genes do not harbor a perfect match to this consensus sequence, but rather contain one or more degenerate motifs. The differences in these motifs may reflect different promoter architectures e.g. AT content , modes of action e.g.", "The differences in these motifs may reflect different promoter architectures e.g. AT content , modes of action e.g. activation or repression or levels of regulation. Spo0A genes in B. subtilis can be divided in different classes that respond to different levels of phosphorylated Spo0A . For C. difficile, we conclude that the Spo0A protein likely recognizes a motif that is similar to the B. subtilis Spo0A box on the basis of four lines of evidence; 1. All DNA binding/contacting residues are conserved Fig. 2B , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A Fig. 2D , 3.", "2B , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A Fig. 2D , 3. Mutagenesis of key residues in the B. subtilis Spo0A box reduces affinity of C. difficile Spo0A for DNA Fig. 2E and 4. A B. subtilis Spo0A box has predictive value for DNA binding by C. difficile Spo0A Fig. 3A . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein.", "3A . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein. It is likely that differences do exist between the preferred binding sites for both proteins that will be evident when a comprehensive analysis is performed of in vivo DNA binding of C. difficile Spo0A; based on the limited data set of this study, a MEME analysis already suggests possible differences in the extended Spo0A motif W.K. Smits, unpublished observations . These differences may relate to the much higher AT content of C. difficile compared to B. subtilis 71 vs. 56.5%, respectively , or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation for review see ref .", "These differences may relate to the much higher AT content of C. difficile compared to B. subtilis 71 vs. 56.5%, respectively , or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation for review see ref . The activation of Spo0A is controlled by a multi-component phosphorelay that can integrate environmental cues and ensures a gradual increase in the level of phosphorylated Spo0A in the cell . In addition, the transcription of the spo0A gene is controlled by multiple feedback loops. For instance, Spo0A regulates its own transcription by binding to the spo0A promoter , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter . In C. difficile, there are some interesting differences and similarities in the regulatory pathways.", "For instance, Spo0A regulates its own transcription by binding to the spo0A promoter , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter . In C. difficile, there are some interesting differences and similarities in the regulatory pathways. Most notably, there seems to be no phosphorelay and the phosphorylation state of Spo0A is supposedly controlled by orphan histidine kinases . The transcription of spo0A in C. difficile is under control of the transition state sigma factor Sigma H , as it is in B. subtilis . Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile Fig. 3A , raising the possibility of auto-regulation of spo0A.", "Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile Fig. 3A , raising the possibility of auto-regulation of spo0A. The putative direct regulation of sigH by Spo0A may reflect that the C. difficile genome does not harbor a homolog of the pleiotropic regulator AbrB, which is responsible for the Spo0A-dependent regulation of sigH in B. subtilis . Consistent with a model in which spo0A is positively autoregulated, we noted a sharp increase in the levels of Spo0A as cells approach the stationary growth phase Figure 1C . Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA Fig. 3B .", "Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA Fig. 3B . All these observations are consistent with direct regulation of these genes by Spo0A in other organisms , and the conservation of the sporulation pathway . Though Spo0A is the key regulator for sporulation in Firmicutes, it regulates numerous other processes in various bacteria. In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology 10, . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB.", "In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology 10, . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB. Additionally, transcription of abrB responds already to low levels of Spo0A,P . As a result these effects are detectable in late-exponential and early stationary phase, as some Spo0A is present throughout growth in B. subtilis cells. Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect.", "Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect. In Clostridium acetobutylicum and C. beijerinckii, Spo0A is a direct regulator of solvent formation, as well as sporulation . It seems therefore conceivable that Spo0A in C. difficile also affects aspects of metabolism. In this respect, it is important to note that also in C. difficile Spo0A is detectable from early exponential growth phase on Figure 1B . We observed direct binding of C. difficile Spo0A to the promoter region of sigH Fig. 3A .", "We observed direct binding of C. difficile Spo0A to the promoter region of sigH Fig. 3A . This gene encodes the key sigma factor for the transition phase, and regulates processes outside sporulation as well . Moreover, we found significant levels of Spo0A from early stationary phase on Fig. 1B and unpublished observations , indicating the regulatory actions of Spo0A need not be limited to stationary phase in C. difficile. In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA Fig. 3A .", "In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA Fig. 3A . The presence of a putative Spo0A binding site upstream of these genes, as well as the spacing compared to the start codon, is conserved in the problematic Stoke-Mandeville strain R20291 , a member of PCR ribotype 27. This could indicate that these aspects of regulation by Spo0A are conserved in multiple strains of C. difficile. It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed.", "It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed. In order to distinguish direct from indirect effects, in vivo binding profiles of Spo0A should be performed. The antibodies generated for this study should prove to be useful for this type of experiments. Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control .", "Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control . Similarly, the production of the emetic toxin cereulide in B. cereus is greatly repressed in a spo0A mutant, in an AbrB-dependent manner . In contrast, Spo0A directly represses the expression of the cry toxin genes in B. thuringiensis and a spo0A mutant is therefore a hyper-producer of the insecticidal crystal protein . In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A and also the production of enterotoxin in this organism seems to be indirectly dependent on sporulation . In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A TcdA , both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B TcdB .", "In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A and also the production of enterotoxin in this organism seems to be indirectly dependent on sporulation . In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A TcdA , both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B TcdB . Our in vitro binding data indicate a potential binding site for Spo0A upstream of tcdB and possibly tcdC Fig. 4A . However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant a kind gift of the Minton lab; did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type #2fold in exponential phase cells up to 4-fold in late-stationary phase cells .", "However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant a kind gift of the Minton lab; did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type #2fold in exponential phase cells up to 4-fold in late-stationary phase cells . The small, and not significant, differences in toxin levels in our experiments might be attributed to differences in the susceptibility of cells for lysis rather than the production of toxin, but could also indicate a negative regulatory effect of Spo0A on toxin production. In support of the latter hypothesis, it was recently reported that a spo0A mutant of C. difficile strain R20291 a PCR ribotypes 027/BI/NAP1 epidemic strain demonstrates ,10fold higher toxin levels than its isogenic wild type 30 h post inoculation, and is significantly more virulent in a mouse model of disease . The differences between Underwood et al on the one hand and our study as well as the study of Deakin and coworkers on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously data not shown .", "The differences between Underwood et al on the one hand and our study as well as the study of Deakin and coworkers on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously data not shown . Alternatively, the differences could indicate integration of the group II intron at more than one location in the chromosome in the strain used in Underwood et al . In the absence of a complementation experiment and/or Southern blot data, this remains to be established. In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays.", "In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays. In the present study we have for the first time demonstrated direct binding of the DNA binding domain of C. difficile Spo0A to putative target DNA. This work has revealed that aspects of Spo0A binding are conserved between Bacillus and C. difficile 0A box, possible auto-regulation and binding to early sporulation promoters , whereas others are not the absence of abrB as a direct target in C. difficile, binding to DNA upstream of lplA, ssuA . The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo.", "The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo. The identification of genes affected by Spo0A in C. difficile may shed light on the role of the protein in virulence and pathogenesis of this organism. Figure S1 Specificity controls for binding by Spo0A-DBD-his6. Arrows indicate the position of shifted species DNA:protein complexes . Titrations with PCR fragments of PabrB containing a high affinity binding site and PtcdA lacking such a site correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A.", "Titrations with PCR fragments of PabrB containing a high affinity binding site and PtcdA lacking such a site correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A. Comparison of binding of Spo0A-DBD-his6, Spo0A-his6 and CD2195-his6 binding to the upstream region of spoIIAA. B. Binding of Spo0A-DBD-his6 to the upstream region of spoIIAA is reversed by the addition of PabrB, but not by the addition of PtcdA . C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA.", "C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA. E. Binding of Spo0A-DBD-his6 to the upstream region of tcdB is reversed by the addition of PabrB, but not by the addition of PtcdA. F. Binding of Spo0A-DBD-his6 to the upstream region of tcdC is not or moderately affected by the addition of PabrB and/or PtcdA. TIF Text S1 Oligonucleotides used in this study and PCR cycling conditions for the EMSA probes. PDF" ]

[ "Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection CDI , for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630.", "Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct putative targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds.", "These results identify for the first time direct putative targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely. Text: Sporulation is an adaptive strategy that enables bacteria to survive harsh environmental conditions for prolonged periods of time, and is an integral part of the transmission of sporulating pathogens and their tolerance and resistance towards antimicrobial compounds. Spo0A is the key regulator for sporulation . Most of our knowledge about the protein is based on work in Bacilli. Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay .", "Spo0A is a response regulator that demonstrates phosphorylation dependent binding to DNA . Phosphorylation occurs through the concerted action of several proteins that together form a so called phosphorelay . The signaling cascade allows for the integration of environmental signals into the regulation of Spo0A dependent processes, including sporulation. The two functional domains, the N-terminal phosphorylation and dimerization domain receiver domain , and the C-terminal DNA binding effector domain are separated by a hinge region that is relatively poorly conserved . Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation .", "Phosphorylation is believed to result in a structural rearrangement that facilitates dimerization , resulting in the disruption of transcription-inhibitory contacts between the receiver and effector domains. The isolated DNA binding domain can bind legitimate targets of the Spo0A protein due to the absence of the transcription inhibitory contacts, thereby bypassing the need for phosphorylation . Extensive characterization of Spo0A targets has revealed a motif that represents a high affinity Spo0A binding site, the 0A box . The crystal structure of the DNA binding domain confirms specific and non-specific contacts between the protein and the consensus sequence . It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis , and solvent production in Clostridium acetobutylicum . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection CDI for recent reviews see .", "It is noteworthy that Spo0A regulates many other processes than sporulation, such as competence for genetic transformation, DNA replication, and biofilm formation in B. subtilis , virulence factors and stress responses in for instance B. anthracis and B. thuringiensis , and solvent production in Clostridium acetobutylicum . C. difficile is a Gram positive, anaerobic bacterium that is the causative agent of C. difficile infection CDI for recent reviews see . Though many people are asymptomatically colonized by C. difficile, the bacterium can cause serious health problems, such as pseudomembranous colitis and toxic megacolon, under the influence of risk factors such as age and antibiotic use. As a result, CDI was long regarded a nosocomial infection. Recently, however, an increase in the cases of community acquired CDI can be observed . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 BI/ NAP1 and 078 .", "Recently, however, an increase in the cases of community acquired CDI can be observed . Outbreaks of CDI have been linked to so called hypervirulent strains, such as PCR ribotypes 027 BI/ NAP1 and 078 . Its main virulence factors are the major clostridial toxins A and B . In addition, certain strains of C. difficile, including ribotypes 027 and 078, additionally encode a binary toxin . C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow .", "C. difficile is transmitted via the fecal-oral route. It is believed that spores are crucial to successfully infect new hosts, as they are able to withstand the harsh environment of the stomach, and survive antibiotic treatments that alter the endogenous flora, after which C. difficile can overgrow . There is limited knowledge about the regulation of sporulation in C. difficile. It has been reported that spo0A, as expected, is required for the formation of spores and the gene is required for persistence and transmission in mice . Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A .", "Though the pathways downstream of Spo0A seem to a large extent conserved between B. subtilis and Clostridia, this is less so for the pathways leading to activation of Spo0A . It has been suggested that the orphan histidine kinase CD2492 is involved in the activation of Spo0A . Similarly, it was reported that multiple orphan histidine kinases can phosphorylate Spo0A in C. acetobutylicum . Recently, it was reported that spo0A can be transcribed from a SigH-dependent promoter . It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes.", "It is unknown which genes are regulated by direct binding of Spo0A to their upstream regions. Here, we establish an in vitro binding assay for C. difficile Spo0A and demonstrate for the first time direct binding of this transcription factor to DNA upstream of several putative target genes. Escherichia coli strains were routinely grown in Luria-Bertani broth or plates, supplemented with appropriate antibiotics. Chloramphenicol was used at a final concentration of 20 mg/mL for agar plates and 10 mg/mL for liquid cultures. Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL.", "Ampicillin was used at a final concentration of 100 mg/mL. Kanamycin was used at a final concentration of 20 mg/mL. Cloning was carried out using E. coli DH5a, overexpression was performed in E. coli Rosetta DE3 pLysS Novagen . C. difficile strains were grown in a glucose-free trypton-yeast based medium TTY; 3% w/v bactotrypton BD , 2% yeast extract Fluka , 0.1% w/v thioglycollate Sigma pH 7.4 , supplemented with 20 mg/mL of lincomycin when appropriate, or on CLO or TSS plates Biomerieux . All plasmids are listed in Table 1 . Primers obtained from Sigma Aldrich are listed in Text S1 and specific cycling conditions are available on request.", "All plasmids are listed in Table 1 . Primers obtained from Sigma Aldrich are listed in Text S1 and specific cycling conditions are available on request. Unless noted otherwise, PCR reactions were carried out using Pfu polymerase Fermentas according to the instructions of the manufacturer. Plasmid pWKS1251, for the overproduction of Spo0A-DBD carrying a C-terminal 66His-tag, was constructed as follows. A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO Invitrogen , yielding pWKS1247.", "A sequence corresponding to the DNA binding domain of Spo0A was amplified using primers oWKS-1123a and oWKS-1124 using chromosomal DNA from C. difficile strain 630Derm as a template. The resulting fragment was cloned into pCR2.1-TOPO Invitrogen , yielding pWKS1247. This plasmid was digested with NdeI and XhoI, separated on a 1% agarose/0.56 TAE 20 mM Tris Acetate, 0.5 mM EDTA gel, the fragment corresponding to the DNA binding domain was recovered by gel-isolation using a GeneJET Gel Extraction kit, Fermentas and cloned into similarly digested pMF14 that had been gel-isolated in the same manner. The construct was verified by PCR, restriction analyses and DNA sequencing using primers oWKS-135 and oWKS-136 see below . Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO.", "Plasmid pWKS1245, for the production of full length Spo0A carrying a C-terminal 6xHis-tag, was constructed in a similar manner using chromosomal DNA from C. difficile 630Derm as a template, but using the PCR product of primers oWKS-1122 and oWKS-1123a. Plasmids used as PCR templates for generating EMSA probes were constructed by cloning the PCR products into pCR2.1-TOPO. The inserts, and in the case of the mutated PabrB promoters the presence of the desired point mutations in the consensus 0A box, were verified by DNA sequencing using primers oWKS-24 and oWKS-25 see below . Sequence grade plasmids were isolated using a Nucleospin Plasmid QuickPure kit Macherey Nagel according to the manufacturer's instructions, except that two lysis reactions were combined onto a single filter and eluted with 65uC prewarmed AE buffer. All constructs were sequenced using BigDye Terminator chemistry Invitrogen on an ABI3130 sequencer Perkin Elmer , according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix Invitrogen in a final volume of 20 mL.", "All constructs were sequenced using BigDye Terminator chemistry Invitrogen on an ABI3130 sequencer Perkin Elmer , according to the instructions of the manufacturers. In short, ,200 ng of plasmid was mixed with 3.2 pmol of primer, 1 mL Terminator Ready Reaction Mix Invitrogen in a final volume of 20 mL. After thermocycling, DNA was precipitated and washed with 65% isopropanol, and dissolved in 12 mL HiDi formamid Invitrogen at 96uC for 2 mins and stored in the dark at 4uC until the sequencing run. Sequence analyses were performed in CloneManager Professional Suite 7 SciEd and Geneious version 5.6.2 Biomatters Ltd . Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta DE3 pLysS Novagen . Transformants were used to inoculate 25 mL of LB with appropriate antibiotics.", "Plasmids pWKS1245 and pWKS1251 were transformed into E. coli Rosetta DE3 pLysS Novagen . Transformants were used to inoculate 25 mL of LB with appropriate antibiotics. After overnight incubation, the cells were 1:100 diluted in 500 mL fresh medium containing appropriate antibiotics. Protein production was induced with 1 mM IPTG at an OD600 of 0.7 and growth was continued for another three hours before harvesting. Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described .", "Cells were washed with ice cold PBS and stored at 280uC for later use. Purification of the proteins was essentially done as described . In short, cells were disrupted in 4 mL lysis buffer 2 mM PMSF, 10 mM imidazole, 5 mM beta-mercaptoethanol, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . Cleared cell lysates we incubated with 2 mL pre-equilibrated 50% TALON slurry Clontech in a final volume of 15 mL lysis buffer for 1 hr. The resin was allowed to settle on a Poly-Prep column BioRad and washed with 2 mL wash buffer 20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column identical to wash buffer but with 50, 100, 250 or 500 mM imidazole .", "The resin was allowed to settle on a Poly-Prep column BioRad and washed with 2 mL wash buffer 20 mM imidazole, 300 mM NaCl, 50 mM NaH 2 PO 4 , pH 7.9 . The protein was stepwise eluted in 1 mL fractions after applying 2 mL elution buffer to the column identical to wash buffer but with 50, 100, 250 or 500 mM imidazole . The whole procedure was carried out at 4uC. Fractions were assayed for purity and yield and suitable fractions were dialysed against 26 1L dialysis buffer 50 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM DTT using Slide-A-Lyzer cassettes with a molecular weight cut-off of 3.5 kDa Pierce . Proteins were stored at 280uC in storage buffer identical to dialysis buffer but containing 20% glycerol . Protein concentrations were determined using Bradford reagent BioRad , according to the manufacturer's instructions.", "Proteins were stored at 280uC in storage buffer identical to dialysis buffer but containing 20% glycerol . Protein concentrations were determined using Bradford reagent BioRad , according to the manufacturer's instructions. DNA fragments for use in EMSA experiment were generated by PCR using GoTaq polymerase Promega and chromosomal DNA from B. subtilis JH642 Bacillus Genetic Stock Center 1A96; plasmids listed in Table 1 , or chromosomal DNA from C. difficile 630Derm as a template. Primers and specific cycling conditions for generation of the EMSA probes are listed in Text S1. DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS and a QIAExII kit Qiagen , according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase Invitrogen according to the instructions of the manufacturer.", "DNA fragments of the expected size were isolated from a 16TAE/8% native polyacrylamide gel using diffusion buffer 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8, 0.1% SDS and a QIAExII kit Qiagen , according to the manufacturer's instructions. Recovered DNA was end-labeled with 32P-c-ATP using FR buffer and T4 kinase Invitrogen according to the instructions of the manufacturer. Specific activity was determined on a LS6000 scintillation counter Beckman . EMSA conditions were based on previous studies . In short, binding reactions were carried out in binding buffer 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol in the presence of 200 mg/mL bovine serum albumin NEB and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer.", "In short, binding reactions were carried out in binding buffer 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 5% glycerol in the presence of 200 mg/mL bovine serum albumin NEB and 200 cpm/mL radiolabeled DNA fragment. Reactions were incubated for 20 minutes at 30uC prior to loading on a 16TAE/8% non-denaturing polyacrylamide gel that was prerun for 20 minutes at 50 V in 16 TAE buffer. Electrophoresis was carried out for 120 min at 85 V. After vacuum drying the gels onto filter paper, they were imaged after overnight exposure on Phosphorimager screens on a Typhoon instrument GE Healthcare . The toxic effects of C. difficile culture supernatants on Vero cells a kind gift of Eric Snijder were determined as follows. Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium Dulbecco modified Eagle medium Lonza supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum , before applying them to a monolayer of Vero cells, and incubation was continued for another hour.", "Supernatant from a bacterial culture was harvested by centrifuging cells for 3 minutes at 140006g and filtered on a 0.45 mM cellulose acetate filter using a syringe. Supernatants were 2-fold serially diluted in cell culture medium Dulbecco modified Eagle medium Lonza supplemented with 100 mg/mL penicillin, 100 U/mL streptomycin, 10% fetal calf serum , before applying them to a monolayer of Vero cells, and incubation was continued for another hour. As a positive control, 50 mL 1:10 diluted purified toxin Techlab was added to the cells. To determine if observed cytotoxic effects were specific for the large clostridial toxins, commercially available anti-toxin against TcdA and TcdB Techlab was added to 10-fold diluted bacterial supernatant for 60 min prior to incubation on the Vero cells. Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects cell rounding were observed. Statistical significance was evaluated with an independent sample t-test.", "Toxin end-point titres were defined as the lowest dilution at which no cytopathological effects cell rounding were observed. Statistical significance was evaluated with an independent sample t-test. Immunization of mice with full length C. difficile Spo0A-6xHis was kindly performed at the Welcome Trust Sanger Institute Hinxton, UK . Cells from 1 mL of C. difficile culture were collected by centrifugation for 1 min at 14000 rpm in a table top centrifuge and resuspended in 200 mL resuspension buffer 10 mM Tris HCl pH 8, 10 mM EDTA, 0.5 mg/mL lysozyme, 1 mM Pefabloc SC Roche . After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer 0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB was added, and samples were heated to 96uC for 5 mins. Total cell lysates amounts corrected for OD 600 were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride PVDF membrane.", "After incubation for 30 mins at 37uC, 50 mL of 56 SDS sample buffer 0.1 M DTT, 2% SDS, 50 mM Tris HCl pH 6.8, 10% glycerol, 0.0025% BPB was added, and samples were heated to 96uC for 5 mins. Total cell lysates amounts corrected for OD 600 were separated on a 12% SDS-PAGE gel prior to semi-dry blotting for 1 h at 10 V to a polyvinylidene fluoride PVDF membrane. Membranes were blocked in PBST buffer phosphate buffered saline with 0.1% v/v Tween-20 containing 5% membrane blocking reagent Amersham Biosciences . To visualize Spo0A protein cleared polyclonal serum from a single mouse at a 1:3000 dilution was used, followed by either a goat-anti-mouse HRP-conjugated secondary antibody followed by ECL+ detection Amersham Bioscience , or a goatanti-mouse-biotin-conjugated secondary antibody Dako followed by a tertiary mouse-anti-biotin Cy3-conjugated antibody Jackson . Detection was done using on a Typhoon instrument GE Healthcare . Background corrected peak volumes were quantified using ImageQuant TL Amersham Biosciences .", "Detection was done using on a Typhoon instrument GE Healthcare . Background corrected peak volumes were quantified using ImageQuant TL Amersham Biosciences . Alignments of B. subtilis and C. difficile spo0A were made using ClustalW2 on the basis of the published genome sequences, Genbank accession numbers AL009126 and AM180355, respectively, and the 630Derm spo0A sequence as determined in this study. The sequence for spo0A of C. difficile strain 630Derm was deposited in Genbank accession no JX050222 . Consensus Spo0A boxes were identified using a Single string Search command in Genome2D , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel.", "Consensus Spo0A boxes were identified using a Single string Search command in Genome2D , allowing 0 mismatches. The box positions were linked to upand downstream genes using the ''Add nearest gene to List of DNA Motifs'' feature and Microsoft Excel. The results were manually inspected for those boxes within 500 bp upstream of a gene on the same strand. Figures for publication were prepared using ImageQuant TL Amersham Biosciences , Adobe Photoshop CS3 Adobe Systems Inc and Corel Graphics Suite X5 Corel Corporation . In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain DBD were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli Fig. 1A and purified to near homogeneity using metal affinity chromatography Fig.", "In order to characterize C. difficile Spo0A, the full length protein and its DNA binding domain DBD were expressed as a Cterminally 66His-tagged protein in the heterologous host Escherichia coli Fig. 1A and purified to near homogeneity using metal affinity chromatography Fig. 1A ; lanes P . Full length protein was used to raise antibodies to detect Spo0A in total lysates of C. difficile strains, and the purified DNA binding domain was used in subsequent in vitro binding assays see below . We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells.", "We determined the expression of C. difficile Spo0A throughout growth. We found that the protein is present in lysates from exponential to stationary growth phase cells. We performed immunoblotting using polyclonal antibodies against C. difficile Spo0A on total lysates of wild type and spo0A mutant cells grown in a trypton-yeast based medium TTY . We found a clear signal of the size expected for full length Spo0A ,31 kDa as early as 3 hours post inoculation exponential growth phase , through transition phase 8 h as well as 24 and 48 hours post inoculation stationary growth phase Figure 1B; 630Derm . The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant Fig. 1B , CT::spo0A .", "The signals were specific for C. difficile Spo0A as they were absent from lysates from the C. difficile spo0A mutant Fig. 1B , CT::spo0A . We obtained similar results in other media, such as the commonly used supplemented brain heart infusion broth BHIS; data not shown . To determine relative levels of Spo0A throughout growth, we performed an immunoblot experiment using fluorescent antibodies, which gives more quantitative information compared to the use of horseradish peroxidase conjugated antibodies in our hands. We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation Figure 1C . Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase.", "We found that the levels of Spo0A increases approximately 20-fold from 6 hours post inoculation and remains at similar levels from 8 to 48 hours post inoculation Figure 1C . Though it should be noted that the Western blots do not provide information on the phosphorylation state of the protein, we conclude that the protein in active or inactive form is present throughout growth and is more abundant in stationary growth phase. Spo0A of C. difficile Strain 630Derm Contains a 6aminoacid Duplication BLAST homology searches readily identify a homolog of the well-characterized B. subtilis Spo0A protein in C. difficile 630 CD1214 and previous work demonstrated that a spo0A mutant an insertional inactivation of cd1214 -as expected -no longer forms spores . In silico analyses suggest a similar secondary structure for both proteins Fig. 2A , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region . We compared the sequence of CD1214 obtained from our lab strain 630Derm to that of the published C. difficile 630 genome .", "2A , with a conserved dimerization and DNA binding domain, separated by a poorly conserved hinge region . We compared the sequence of CD1214 obtained from our lab strain 630Derm to that of the published C. difficile 630 genome . Strain 630Derm is a spontaneous erythromycin sensitive strain, which is commonly used in mutagenesis studies and was obtained by serial passaging of strain 630 . The 630Derm spo0A sequence Genbank accession no JX050222 was derived from the expression plasmids constructed for this study, and confirmed in a whole genome sequence of strain 630Derm generated in our lab data not shown . We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid NVGNIE duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation hinge , flanking the highly conserved DNA binding domain Fig.", "We found that 630Derm spo0A contains an 18 base pair direct repeat, resulting in a 6 amino acid NVGNIE duplication compared to the published reference sequence. The duplication maps to a region of the protein with relatively low sequence conservation hinge , flanking the highly conserved DNA binding domain Fig. 2A and B . We verified the absence of this duplication in strain 630 by PCR Fig. 2C as well as sequencing from the chromosomal DNA of C. difficile 630 data not shown , to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 to which 630 and 630Derm belong by PCR, but the duplication was found to be unique to 630Derm among the isolates tested data not shown .", "2C as well as sequencing from the chromosomal DNA of C. difficile 630 data not shown , to rule out an error in the original genome sequence and to demonstrate that the difference in size of the PCR product was specific to the 18 bp insertion. In addition, we checked several other strains of PCR ribotypes 12 to which 630 and 630Derm belong by PCR, but the duplication was found to be unique to 630Derm among the isolates tested data not shown . C. difficile Spo0A-DBD Shows Similar Specificity as B. subtilis Spo0A-DBD Next, we examined the conservation of the DNA binding domain of Spo0A Spo0A-DBD between B. subtilis and C. difficile. In B. subtilis amino acid residues contacting the backbone of the DNA and interacting with specific residues of the Spo0A binding sequence have been defined . We found that all these residues were conserved in the C. difficile protein sequence Fig. 2B , indicating that the protein likely recognizes a similar motif.", "We found that all these residues were conserved in the C. difficile protein sequence Fig. 2B , indicating that the protein likely recognizes a similar motif. DNA binding by full length Spo0A in B. subtilis requires phosphorylation dependent dimerization . However, it was shown that the isolated DBD is capable of binding to legitimate targets of the full length protein . Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene PabrB of B. subtilis.", "Analogously, we purified the C. difficile Spo0A-DBD for use in in vitro binding assays. As no direct targets for the C. difficile protein have been reported so far, we used the upstream region of the abrB gene PabrB of B. subtilis. PabrB is commonly used as a high-affinity control in binding assays with the B. subtilis Spo0A or Spo0A-DBD protein . It is noteworthy that we failed to identify a homolog of abrB in C. difficile using BLAST, indicating that potential indirect regulation by Spo0A cannot occur through abrB in C. difficile as it does in B. subtilis. We found that C. difficile Spo0A-DBD bound with high affinity to PabrB Fig. 2D and E .", "We found that C. difficile Spo0A-DBD bound with high affinity to PabrB Fig. 2D and E . We performed electrophoretic mobility shift assays EMSAs using radiolabeled PabrB and increasing amounts of purified C. difficile Spo0A-DBD that was purified using a C-terminal 66His-tag. The addition of protein leads to a dose-dependent retardation of the DNA fragment with an apparent K D of ,50 nM. In the same range of protein concentrations, no binding was observed for a negative control a DNA fragment of B. subtilis citG Fig. 2E , suggesting that binding was specific for the abrB promoter region. B. subtilis Spo0A recognizes a distinct sequence 0A box , that is characterized by a 7 bp core motif TGTCGAA .", "2E , suggesting that binding was specific for the abrB promoter region. B. subtilis Spo0A recognizes a distinct sequence 0A box , that is characterized by a 7 bp core motif TGTCGAA . Structural studies have revealed that the protein makes specific contacts with the G at position 2 G2 , and the C at position 4 C4 and 5 G5 of this motif . We introduced G2A, C4A, G5A, G2A/C4A and C4A/G5A mutations in the perfect consensus core 0A-box present in PabrB. We found that the affinity of C. difficile Spo0A for these mutated PabrB fragments was highly reduced Fig. 2E . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence.", "2E . We performed EMSAs using radiolabeled PabrB containing the mutated core sequence. For the single point mutations in the DNA, the affinity decreased ,10-fold. There did not seem to be an additive effect of a second point mutation for the two combinations tested. None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other non-consensus 0A boxes in the abrB promoter . Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD.", "None of the mutations abolished binding of C. difficile Spo0A completely, most likely as the result of binding of Spo0A to other non-consensus 0A boxes in the abrB promoter . Taken together, we conclude that the guanine and cytosine residues in the core TGTCGAA motif of PabrB are important for specific binding of this fragment by C. difficile Spo0A-DBD. Value for Binding by C. difficile Spo0A-DBD Above, we have established that the Spo0A-DBD of C. difficile is highly homologous to that of the B. subtilis Spo0A protein, and that the proteins recognize a similar consensus sequence Fig. 2 . Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A. We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D .", "Based on this information, we identified the several genes as putative direct targets of C. difficile Spo0A. We queried the C. difficile 630 genome sequence for perfect matches to the core 0A box using Genome2D . Such an analysis revealed the presence of 102 matching motifs, of which 45 were located within 500 bp of the initiating ATG of an open reading frame on the same strand see Table S1 . Our attention was drawn to spo0A and sigH, as these two genes were previously found to be regulated by Spo0A in B. subtilis and/or play important roles in sporulation 3, . We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH. We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames.", "We found that C. difficile Spo0A bound to DNA sequences upstream of spo0A and sigH. We performed EMSAs with DNA encompassing 220-281 bp upstream of the initiating ATG codon of the spo0A, sigH and spoVG open reading frames. We found that the addition of Spo0A-DBD to the reactions caused retardation of the spo0A and sigH DNA fragments Fig. 3A , but not of a spoVG fragment which did not contain a consensus 0A box Fig. 3B . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA.", "3B . It should be noted that the affinity of Spo0A-DBD for the region upstream of spo0A was the highest we have observed so far for any C. difficile DNA. Moreover, the presence of multiple shifted species could indicate the presence of more than one strong binding site. These results establish that spo0A and sigH are likely legitimate targets of Spo0A in C. difficile, and confirm that spoVG is not, in line with results obtained in B. subtilis . We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon.", "We were interested to see if Spo0A in C. difficile could potentially regulate genes that have no documented function in sporulation. Our in silico analysis identified several genes with no obvious link to sporulation that had a consensus 0A box within 100 bp upstream of their start codon. This positioning is similar to that observed for spo0A . and sigH .. We confirmed in vitro binding of the C. difficile Spo0A-DBD to the promoter regions of lplA and ssuA. We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene CD1654; box at 267 and the ssuA gene CD1484; box at 282 Fig.", "We carried out EMSA experiments using probes that included the perfect consensus site and purified Spo0A-DBD protein. We observed binding of the protein to fragments upstream of the lplA gene CD1654; box at 267 and the ssuA gene CD1484; box at 282 Fig. 3A . The lplA gene encodes a predicted lipoate-protein ligase, and ssuA is annotated as an aliphatic sulfonates ABC transporter; to our knowledge, neither of these have been directly implicated in sporulation or have found to be targets for Spo0A in other organisms. Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile. It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway .", "Together our results establish the potential for binding of Spo0A to DNA upstream of spo0A and sigH, two genes that are important for sporulation, and indicate that Spo0A may have functions that go beyond the regulation of sporulation in C. difficile. It has been established that a spo0A mutant of C. difficile does not produce any spores, consistent with a crucial role in the sporulation pathway . However, the in silico identification of upstream regions with a consensus Spo0A binding site did not point to any of the early sporulation genes downstream of spo0A itself as direct targets of Spo0A. This is likely the result of variations in the 0A-box in these promoters that were disregarded in the box search. In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A such as spoIIAA and spoIIE do not contain a 100% match to the core motif, but rather one or more near-consensus boxes . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above Fig.", "In support of this, many well-characterized legitimate direct targets of B. subtilis Spo0A such as spoIIAA and spoIIE do not contain a 100% match to the core motif, but rather one or more near-consensus boxes . We found that Spo0A- We performed EMSA experiments using increasing amounts of purified Spo0A-DBD from C. difficile 630Derm and the DNA fragments indicated above Fig. 3B . For spoIIAA encoding an antianti sigma-factor and spoIIE encoding a serine phosphatase , we observed a low intensity shifted species at concentrations as low as 150 nM. For spoIIGA encoding a sporulation specific protease we observed the shifted species only at higher concentrations of protein .200 nM . The negative control spoVG did not demonstrate binding of Spo0A-DBD at these concentrations.", "For spoIIGA encoding a sporulation specific protease we observed the shifted species only at higher concentrations of protein .200 nM . The negative control spoVG did not demonstrate binding of Spo0A-DBD at these concentrations. Moreover, the shift we observed was reversible using unlabeled DNA containing a high affinity binding site, but not using unlabeled DNA that lacked such a site Figure S1B-D . Therefore, we consider the binding to spoIIAA, spoIIE and spoIIGA genes to be specific, despite the fact that increasing the amount of protein did not seem to cause a significant increase in the amount of DNA in the complex. Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions. C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells .", "Together, these results suggest that Spo0A in C. difficile might regulate the transcription of at least a subset of early sporulation genes by direct binding to their promoter regions. C. difficile Spo0A-DBD Binds to DNA Upstream of tcdB It has previously been reported that the deletion of Spo0A in C. difficile results in a significantly lower toxin production and a ,1000-fold reduction in the toxicity of culture supernatant derived from spo0A negative cells towards Vero cells . Considering the absence of a homolog of the abrB repressor, direct binding of Spo0A and concomitant activation of toxin gene transcription is a likely mechanism through which this could occur. We found evidence for direct binding of Spo0A-DBD to the region upstream of tcdB, encoding one of the major clostridial toxin genes, and possibly tcdC, but this did not seem to result in lower toxin levels in our hands. We performed EMSAs using DNA upstream of tcdR encoding a sigma factor responsible for the activation of toxin gene transcription , tcdB encoding toxin B , tcdA encoding toxin A . In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE encoding a holin-like protein and tcdC encoding a putative negative regulator of toxin production , even though this regulator does not have a significant effect on toxin levels under the conditions we used .", "We performed EMSAs using DNA upstream of tcdR encoding a sigma factor responsible for the activation of toxin gene transcription , tcdB encoding toxin B , tcdA encoding toxin A . In order to test regions upstream of all open reading frames in the PaLoc, we also tested binding of Spo0A to DNA upstream of tcdE encoding a holin-like protein and tcdC encoding a putative negative regulator of toxin production , even though this regulator does not have a significant effect on toxin levels under the conditions we used . Of the regions tested, we only observed a clear shifted species, indicative of Spo0A binding, for tcdB Figure 4A ; the shifted species in our EMSA assay was reversed by the addition of unlabeled DNA containing a high affinity binding site, but not by DNA lacking such a site Figure S1E . For tcdC, some smearing was observed at all concentrations of proteins tested Figure 4A , and there did not seem to be a clear effect of the addition of unlabeled DNA fragments Figure S1F . The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG. We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested.", "The probes for tcdA, tcdE and tcdR were indistinguishable from those obtained with our negative control, spoVG. We wanted to determine if toxin levels in culture supernatants were directly or indirectly affected by Spo0A, as was previously suggested. We found no lower toxicity towards Vero cells of culture supernatants derived from spo0A mutant cells compared to wild type. We grew three independent biological replicates of a wild type 630Derm or Clostron-generated spo0A mutant CT::spo0A -a kind gift of the Minton lab in glucose-free TTY medium. We harvested culture supernatant at late-exponential phase approximately 7 hours post inoculation , the transition phase between exponential and stationary growth phase approximately 9 hours post inoculation , as well as two time points in stationary phase 24 and 48 hours post inoculation and determined the toxin endpoint titres see Materials and Methods . In contrast to previous findings, we observed a small #4-fold increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant p.0.05, independent sample t-test .", "We harvested culture supernatant at late-exponential phase approximately 7 hours post inoculation , the transition phase between exponential and stationary growth phase approximately 9 hours post inoculation , as well as two time points in stationary phase 24 and 48 hours post inoculation and determined the toxin endpoint titres see Materials and Methods . In contrast to previous findings, we observed a small #4-fold increase in the toxicity of supernatants derived from spo0A mutant cells compared to wild type, but in all cases this difference was not statistically significant p.0.05, independent sample t-test . In other medium BHIS , we observed no differences at all data not shown . We conclude that Spo0A does not positively affect toxin production in C. difficile 630Derm and the in vivo relevance of the binding to regions upstream of tcdB and/or tcdC is therefore limited under our experimental conditions. The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box .", "The Spo0A-box of C. difficile In B. subtilis, the binding site of Spo0A on target DNA has been well-characterized, through a combination of in vitro binding assays, determination of in vivo binding profiles and mutagenesis of regulated promoter sequences. This work has led to the identification of a conserved core motif, TGTCGAA, or Spo0A box . Depending on the analysis, this motif is flanked by one or more adenine or thymine residues . Interestingly, many target genes do not harbor a perfect match to this consensus sequence, but rather contain one or more degenerate motifs. The differences in these motifs may reflect different promoter architectures e.g. AT content , modes of action e.g.", "The differences in these motifs may reflect different promoter architectures e.g. AT content , modes of action e.g. activation or repression or levels of regulation. Spo0A genes in B. subtilis can be divided in different classes that respond to different levels of phosphorylated Spo0A . For C. difficile, we conclude that the Spo0A protein likely recognizes a motif that is similar to the B. subtilis Spo0A box on the basis of four lines of evidence; 1. All DNA binding/contacting residues are conserved Fig. 2B , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A Fig. 2D , 3.", "2B , 2. C. difficile Spo0A can bind with high affinity to a target of B. subtilis Spo0A Fig. 2D , 3. Mutagenesis of key residues in the B. subtilis Spo0A box reduces affinity of C. difficile Spo0A for DNA Fig. 2E and 4. A B. subtilis Spo0A box has predictive value for DNA binding by C. difficile Spo0A Fig. 3A . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein.", "3A . It is conceivable that our model system, using the purified DNA binding domain, does not accurately reflect binding to all target sites, if target site selectivity is determined in part by other parts by of the full length protein. It is likely that differences do exist between the preferred binding sites for both proteins that will be evident when a comprehensive analysis is performed of in vivo DNA binding of C. difficile Spo0A; based on the limited data set of this study, a MEME analysis already suggests possible differences in the extended Spo0A motif W.K. Smits, unpublished observations . These differences may relate to the much higher AT content of C. difficile compared to B. subtilis 71 vs. 56.5%, respectively , or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation for review see ref .", "These differences may relate to the much higher AT content of C. difficile compared to B. subtilis 71 vs. 56.5%, respectively , or phosphorylation dependent dimerization, for instance. The initiation of sporulation in B. subtilis is subject to complex regulation for review see ref . The activation of Spo0A is controlled by a multi-component phosphorelay that can integrate environmental cues and ensures a gradual increase in the level of phosphorylated Spo0A in the cell . In addition, the transcription of the spo0A gene is controlled by multiple feedback loops. For instance, Spo0A regulates its own transcription by binding to the spo0A promoter , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter . In C. difficile, there are some interesting differences and similarities in the regulatory pathways.", "For instance, Spo0A regulates its own transcription by binding to the spo0A promoter , as well as by indirectly stimulating the transcription of sigH, encoding a sigma factor that recognizes the spo0A promoter . In C. difficile, there are some interesting differences and similarities in the regulatory pathways. Most notably, there seems to be no phosphorelay and the phosphorylation state of Spo0A is supposedly controlled by orphan histidine kinases . The transcription of spo0A in C. difficile is under control of the transition state sigma factor Sigma H , as it is in B. subtilis . Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile Fig. 3A , raising the possibility of auto-regulation of spo0A.", "Our data indicate that both spo0A and sigH could be targets for direct regulation by Spo0A in C. difficile Fig. 3A , raising the possibility of auto-regulation of spo0A. The putative direct regulation of sigH by Spo0A may reflect that the C. difficile genome does not harbor a homolog of the pleiotropic regulator AbrB, which is responsible for the Spo0A-dependent regulation of sigH in B. subtilis . Consistent with a model in which spo0A is positively autoregulated, we noted a sharp increase in the levels of Spo0A as cells approach the stationary growth phase Figure 1C . Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA Fig. 3B .", "Downstream of Spo0A, we found binding of Spo0A to DNA upstream of several early sporulation genes, such as spoIIAA, spoIIE, and spoIIGA Fig. 3B . All these observations are consistent with direct regulation of these genes by Spo0A in other organisms , and the conservation of the sporulation pathway . Though Spo0A is the key regulator for sporulation in Firmicutes, it regulates numerous other processes in various bacteria. In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology 10, . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB.", "In the non-pathogenic B. subtilis, for instance, the protein also affects competence development, biofilm formation, the production of and resistance to antimicrobial compounds, chromosome dynamics and aspects of phage biology 10, . Importantly, several of these processes are indirectly regulated, through the Spo0A-dependent repression of abrB. Additionally, transcription of abrB responds already to low levels of Spo0A,P . As a result these effects are detectable in late-exponential and early stationary phase, as some Spo0A is present throughout growth in B. subtilis cells. Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect.", "Though abrB is absent from C. difficile, this does not exclude the possibility of indirect transcriptional regulation through Spo0Adependent effects on other regulators. Alternatively, Spo0A may exert a direct effect. In Clostridium acetobutylicum and C. beijerinckii, Spo0A is a direct regulator of solvent formation, as well as sporulation . It seems therefore conceivable that Spo0A in C. difficile also affects aspects of metabolism. In this respect, it is important to note that also in C. difficile Spo0A is detectable from early exponential growth phase on Figure 1B . We observed direct binding of C. difficile Spo0A to the promoter region of sigH Fig. 3A .", "We observed direct binding of C. difficile Spo0A to the promoter region of sigH Fig. 3A . This gene encodes the key sigma factor for the transition phase, and regulates processes outside sporulation as well . Moreover, we found significant levels of Spo0A from early stationary phase on Fig. 1B and unpublished observations , indicating the regulatory actions of Spo0A need not be limited to stationary phase in C. difficile. In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA Fig. 3A .", "In line with this idea, we found a potential regulatory link between Spo0A and two genes that to our knowledge are not related to the sporulation process, the lipoate ligase lplA and the aliphatic sulfonates transporter ssuA Fig. 3A . The presence of a putative Spo0A binding site upstream of these genes, as well as the spacing compared to the start codon, is conserved in the problematic Stoke-Mandeville strain R20291 , a member of PCR ribotype 27. This could indicate that these aspects of regulation by Spo0A are conserved in multiple strains of C. difficile. It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed.", "It should be noted that our work so far has been limited to an in vitro analysis of Spo0A binding, and therefore does not indicate whether activation or repression of the putative target genes occurs in vivo. To answer this question, detailed transcriptome and/or proteome studies have to be performed. In order to distinguish direct from indirect effects, in vivo binding profiles of Spo0A should be performed. The antibodies generated for this study should prove to be useful for this type of experiments. Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control .", "Amongst the pathogenic Firmicutes, Spo0A has been reported to affect toxin production in multiple species. In B. anthracis a spo0A mutation results in elevated levels of AbrB, and concomitantly lower levels of the toxin genes pagA, cya and lef that are under AbrB control . Similarly, the production of the emetic toxin cereulide in B. cereus is greatly repressed in a spo0A mutant, in an AbrB-dependent manner . In contrast, Spo0A directly represses the expression of the cry toxin genes in B. thuringiensis and a spo0A mutant is therefore a hyper-producer of the insecticidal crystal protein . In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A and also the production of enterotoxin in this organism seems to be indirectly dependent on sporulation . In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A TcdA , both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B TcdB .", "In Clostridium perfringens TpeL, a member of the large clostridial toxins just like TcdA and TcdB, is directly dependent on Spo0A and also the production of enterotoxin in this organism seems to be indirectly dependent on sporulation . In C. difficile an insertional spo0A mutant generated using Clostron technology was reported to have ,10-fold reduced levels of toxin A TcdA , both intracellularly and extracellularly as well as ,1000-fold reduced toxicity towards Vero cells, which are primarily sensitive towards toxin B TcdB . Our in vitro binding data indicate a potential binding site for Spo0A upstream of tcdB and possibly tcdC Fig. 4A . However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant a kind gift of the Minton lab; did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type #2fold in exponential phase cells up to 4-fold in late-stationary phase cells .", "However, the in vivo relevance of this binding seems limited as in our hands an independently derived but otherwise identical mutant a kind gift of the Minton lab; did not demonstrate a reduced toxicity towards Vero cells. In contrast, we found that in TTY medium toxin levels were slightly elevated in spo0A mutant cells compared to wild type #2fold in exponential phase cells up to 4-fold in late-stationary phase cells . The small, and not significant, differences in toxin levels in our experiments might be attributed to differences in the susceptibility of cells for lysis rather than the production of toxin, but could also indicate a negative regulatory effect of Spo0A on toxin production. In support of the latter hypothesis, it was recently reported that a spo0A mutant of C. difficile strain R20291 a PCR ribotypes 027/BI/NAP1 epidemic strain demonstrates ,10fold higher toxin levels than its isogenic wild type 30 h post inoculation, and is significantly more virulent in a mouse model of disease . The differences between Underwood et al on the one hand and our study as well as the study of Deakin and coworkers on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously data not shown .", "The differences between Underwood et al on the one hand and our study as well as the study of Deakin and coworkers on the other hand may be explained by differences in experimental conditions, such as the medium used. However, we observed no difference in cytotoxicity between supernatant derived from wild type or spo0A mutant cells when they were grown in BHIS, a medium nearly identical to that used previously data not shown . Alternatively, the differences could indicate integration of the group II intron at more than one location in the chromosome in the strain used in Underwood et al . In the absence of a complementation experiment and/or Southern blot data, this remains to be established. In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays.", "In summary, our data are consistent with a model in which the regulation of the major clostridial toxins in C. difficile is not positively affected by Spo0A, in contrast to previous findings and other pathogenic Clostridia. Whether Spo0A is truly a negative regulator of toxin production remains to be confirmed using in vitro and in vivo transcription assays. In the present study we have for the first time demonstrated direct binding of the DNA binding domain of C. difficile Spo0A to putative target DNA. This work has revealed that aspects of Spo0A binding are conserved between Bacillus and C. difficile 0A box, possible auto-regulation and binding to early sporulation promoters , whereas others are not the absence of abrB as a direct target in C. difficile, binding to DNA upstream of lplA, ssuA . The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo.", "The effects of Spo0A on toxin production may be similar to those observed for B. thuringiensis . Future work will be aimed at determining the effect of Spo0A on the transcription of the putative target genes, and carry out a comprehensive analysis of Spo0A binding in vivo. The identification of genes affected by Spo0A in C. difficile may shed light on the role of the protein in virulence and pathogenesis of this organism. Figure S1 Specificity controls for binding by Spo0A-DBD-his6. Arrows indicate the position of shifted species DNA:protein complexes . Titrations with PCR fragments of PabrB containing a high affinity binding site and PtcdA lacking such a site correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A.", "Titrations with PCR fragments of PabrB containing a high affinity binding site and PtcdA lacking such a site correspond to approximately 0.1 nM/mL -0.03 nM/ mL. A. Comparison of binding of Spo0A-DBD-his6, Spo0A-his6 and CD2195-his6 binding to the upstream region of spoIIAA. B. Binding of Spo0A-DBD-his6 to the upstream region of spoIIAA is reversed by the addition of PabrB, but not by the addition of PtcdA . C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA.", "C. Binding of Spo0A-DBD-his6 to the upstream region of spoIIE is reversed by the addition of PabrB, but not by the addition of PtcdA. D. Binding of Spo0A-DBD-his6 to the upstream region of spoIIGA is reversed by the addition of PabrB, but not by the addition of PtcdA. E. Binding of Spo0A-DBD-his6 to the upstream region of tcdB is reversed by the addition of PabrB, but not by the addition of PtcdA. F. Binding of Spo0A-DBD-his6 to the upstream region of tcdC is not or moderately affected by the addition of PabrB and/or PtcdA. TIF Text S1 Oligonucleotides used in this study and PCR cycling conditions for the EMSA probes. PDF" ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Venezuelan equine encephalitis virus VEEV is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing RNA-Seq of poly A and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection.", "Differential expression of interferon response, stress response factors, and components of the unfolded protein response UPR was observed. The protein kinase RNA-like endoplasmic reticulum kinase PERK arm of the UPR was activated, as the expression of both activating transcription factor 4 ATF4 and CHOP DDIT3 , critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 EGR1 was induced in a PERK-dependent manner. EGR1 −/− mouse embryonic fibroblasts MEFs demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans.", "The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes.", "Venezuelan equine encephalitis virus VEEV is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 EGR1 contributed to VEEV-induced cell death. Text: V enezuelan equine encephalitis virus VEEV is a New World alphavirus in the family Togaviridae that is endemic to the Americas. VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . .", "VEEV is a positive-strand RNA virus that is transmitted by mosquitoes and that is naturally present in rodent reservoirs . . There are six subtypes that are categorized by their geographic range and pathology in equines and humans. The two epizootic strains, IA/B and IC, arose from mutations among the enzootic strains . . The IA/B and IC strains are of particular concern due to increased rates of morbidity and mortality and the risks associated with viral amplification and potential species spillover . . In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting.", ". In humans, VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system CNS and neurological symptoms, such as confusion, ataxia, and seizures, manifest. The mortality rate among cases with neurological symptoms can be as high as 35% in children and 10% in adults, with long-term neurological deficits often being seen in survivors . . In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . .", ". In 1995, an outbreak of VEEV in Colombia and Venezuela resulted in over 100,000 human cases . . In addition to natural outbreaks, VEEV is also a concern from a bioterrorism perspective, as it can be grown to high titers, requires a low infectious dose, and contains multiple serotypes. Both the former Soviet Union and the United States previously weaponized the virus, producing large quantities for their now defunct offensive bioweapons programs . . Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations .", "Currently, vaccine strain TC83 is used in horses and for high-risk personnel; however, due to the low rate of seroconversion achieved with this vaccine . and its reliance on two single attenuating mutations . , it is considered unfit for mass distribution . . To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection.", "To date there are no FDA-approved therapeutics for VEEV infection, and further studies are required for clarification of the mechanisms associated with the underlying pathogenesis of VEEV. Viral and host transcriptomic studies can provide a wealth of information on the underlying pathogenic mechanisms and interactions following the course of an infection. The use of highthroughput next-generation sequencing has led to the discovery of previously uncharacterized viruses and the establishment of numerous novel experimental systems redefining virus-host interactions. To date a number of studies have examined the alterations in the host transcriptome following VEEV infection. A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . .", "A comparative microarray analysis between cells persistently infected with VEEV and cells able to clear VEEV resulted in the identification of PARP12L as an antiviral factor . . A molecular comparison utilizing microarrays of host-based responses to the TC83 strain was able to identify biomarkers differentiating between vaccine responder and vaccine nonresponder groups, as well as the involvement of interferon IFN , interferon-induced pathways, Toll-like receptor TLR , and interleukin 12 IL-12 related pathways . . A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . .", "A study examining the role of adhesion and inflammatory factors in VEEV-infected CD-1 mice found viral modulation of the expression of extracellular matrix and adhesion genes, such as integrins Itg␣X, Itg2, 3, and 7 , cadherins 1 and 2, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 ICAM-1 , in the brains of VEEV-infected mice . . Follow-up experiments utilizing ICAM-1-knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of neurological sequelae . . A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II .", "A study by Sharma et al. utilized microarrays to analyze gene expression changes in the brain tissue of VEEV-infected mice over the course of an infection, discovering numerous immune pathways involved in antigen presentation, inflammation, apoptosis, and the traditional antiviral response Cxcl10, CxCl11, Ccl5, Ifr7, Ifi27, Oas1b, Fcerg1, Mif, clusterin, and major histocompatibility complex MHC class II . . A second study by the same group identified the regulation of microRNAs miRNAs in the brains of VEEV-infected mice, which enabled the correlation of the miRNA changes with earlier mRNA expression data . . These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system .", ". These analyses suggest that VEEV may be utilizing cellular miRNAs in order to regulate downstream mRNA, which may correspond with the VEEV-induced histological changes to the nervous system . . In the current study, next-generation RNA sequencing RNA-Seq was used to identify clinically relevant alterations in the mRNA transcriptome of human astrocytes infected with wildtype WT VEEV strain Trinidad donkey TrD . The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts .", "The analysis of host mRNAs by RNA-Seq provides novel insight into how a host responds to a viral infection through the identification of a wide and dynamic range of transcripts in an unbiased manner. Selective sequencing of mRNAs, specifically, polyadenylated poly A transcripts, which account for ϳ1% of the entire transcriptome, enhances the detection of the most relevant and low-abundance transcripts . . As VEEV has been shown to productively infect astrocytes both in vitro and in vivo . , we chose astrocytes as our model of interest. Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain.", "Astrocytes are the most abundant cell in the brain, outnumbering neurons by at least 5-fold . , providing an abundant resource for viral replication within the brain. In addition to their well-described structural role in neuronal tissue, as-trocytes play critical roles in other processes, including the regulation of blood flow and of the blood-brain barrier, synapse transmission, and the response to infection . . VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . .", "VEEV-infected astrocytes have been shown to produce multiple cytokines, including IL-8, IL-17, interferon gamma IFN-␥ , and gamma interferon-induced protein 10, all of which were found to be associated with viral attenuation . . In order to obtain a dynamic view of the virus-host interactome, RNA-Seq was used to monitor changes in gene expression in VEEV TrD-infected astrocytes at 4, 8, and 16 h postinfection hpi . By viewing the alterations at multiple early time points using triplicate biological replicates, a robust and dynamic range of information is generated, and this information provides an increase in both the power and the accuracy of detection of differentially expressed transcripts in a highly relevant clinical model . . Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed.", ". Among VEEV-infected cells, an increase in interferon-regulated genes, including IFIT1, IFIT2, IFIT3, and OASL, was observed. The increased expression of genes involved in the stressinduced unfolded protein response UPR pathway was also noted. Interestingly, VEEV infection resulted in an increase in early growth response protein 1 EGR1 , which may serve as a link between the two pathways. The identification of host mRNAs whose expression is altered following VEEV replication, specifically, EGR1 and its interactors up-and downstream, may provide novel host-based therapeutic targets critical for VEEV replication and a greater understanding of the underlying mechanisms underpinning alphavirus replication. Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources.", "Viral infections and plaque assays. VEEV TrD was obtained from BEI Resources. All experiments with VEEV TrD were performed under biosafety level 3 BSL-3 conditions. All work involving select agents is registered with the Centers for Disease Control and Prevention and was conducted at George Mason University's Biomedical Research Laboratory, which is registered in accordance with federal select agent regulations. For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage.", "For infections, VEEV was added to supplemented Dulbecco modified Eagle medium DMEM to achieve a multiplicity of infection MOI of 0.05, 0.5, or 5. Cells were infected for 1 h at 37°C and rotated every 15 min to ensure adequate coverage. The cells were then washed with phosphatebuffered saline PBS , and complete growth medium was added back to the cells. Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation.", "Viral supernatants and cells were collected at various times postinfection for further analysis. Plaque assays were performed as previously described . . mRNA isolation and poly A library preparation. RNA from U87MG cells was purified from both VEEV TrD-infected biosafety level 3 and mock-infected U87MG cells at 4, 8, and 16 hpi utilizing a mirVana isolation kit Life Technologies . Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit.", "Quality control of purified RNA was then performed using an Agilent 2100 bioanalyzer, and an RNA integrity number RIN cutoff of 8 was utilized for all samples. An External RNA Controls Consortium ERCC RNA spike-in control mix was then added to the total RNA inputs 10 g RNA before poly A selection using a Life Technologies Dynabeads mRNA Direct kit. Preparation of a whole-transcriptome RNA library from purified mRNA was then performed using an Ion Total RNA-Seq kit v2; Life Technologies . Quality control of the cDNA libraries was then performed using the Agilent 2100 bioanalyzer along with sterility testing for removal of libraries for sequencing from a BSL-3 to BSL-2 laboratory. RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies .", "RNA sequencing. Library template preparation was performed on a One Touch 2 platform Life Technologies . Next-generation RNA sequencing was performed on an Ion Torrent PGM platform and was carried out for each sample to assess the differential gene expression of infected versus uninfected cells over time. Data filtering and RNA-Seq analysis pipeline. A total of ϳ119 million sequencing reads and an average of 6.6 million reads per sample were used as the input into our analysis pipeline. Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing.", "Unless otherwise noted, downstream RNA-Seq analysis was carried out using the CLC bio Genomics Workbench v7 . Raw RNA-Seq reads were trimmed to remove any residual sequencing adapter fragments that remained on the 5= or 3= ends after sequencing. In addition, end trimming of reads was done using the modified Mott algorithm with a Q20 quality score, and any reads of less than 15 bp were discarded. Following read trimming, the reads were mapped to human genome hg19 with the following RNA-Seq parameters: a 10-hit limit for multiple mapped positions, a similarity fraction of 0.8, a length fraction of 0.8, a mismatch cost of 2, and an indel cost of 3. The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. .", "The expression level of individual genes and transcripts was calculated using the number of reads per kilobase of the exon model per million mapped reads RPKM method of Mortazavi et al. . . In addition, unmapped reads were also mapped to the ERCC92 synthetic RNA sequence set . , as well as to the VEEV reference genome GenBank accession number L01442 . In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 .", "In all samples, the correlation coefficient R 2 between the expected and the mapped number of reads for the ERCC92 spike-in controls was above 0.90. A summary of the overall sequencing results is shown in Table 1 . Postmapping filtering of all RNA-Seq data was carried out next to include only genes with at least one uniquely mapped read 26,230 genes remained across all data sets and only those with a nonzero interquartile range across the entire experiment. Principal component analysis of the resulting filtered data set 13,906 genes in total was carried out using raw counts of uniquely mapped reads see Fig. 2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff.", "2A . The remaining RPKM expression values for each gene included in the filtered data set were subjected to quantile normalization with a 5% cutoff. A box plot of log 2transformed RPKM values for each sample before normalization is shown in Fig. 2B . The R 2 value for pairwise sample-to-sample variation within each biological replicate set was observed to range from 0.89 to 0.99, indicating that our biological replicates were consistent and showed no strong bias data not shown . Differential gene expression analysis. Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package .", "Differentially expressed genes DEGs were identified using two approaches. First, the empirical analysis of differential gene expression algorithm, part of the edgeR Bioconductor package . , was applied to the integrated data set of all 18 experiments using the default parameters and a false discovery rate-corrected P value. At each time point, infected and mock-infected samples were compared, and genes whose expression differed by more than 2-fold with a significance with a P value of Յ0.05 were provisionally considered to be differentially expressed. In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. .", "In addition to the method described above, an orthogonal statistical test of differential expression was applied to the data using a statistical test developed by Baggerly et al. . to count the number of expressed sequence tags associated with individual genes, a common feature of both serial analysis of gene expression SAGE data and RNA-Seq data. When infected and mock-infected samples were compared, individual genes were provisionally considered differentially expressed when their expression differed by more than 2-fold with a significance with a P value of Յ0.05. Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA.", "Differentially expressed genes found to be in the intersection of the sets of genes identified by both of the methods outlined above were considered high-quality candidates and used as the starting point for further investigation. Clustering and GSEA. Filtered, normalized expression data were subjected to k-means clustering using a Euclidian distance metric where genes were grouped by means of normalized gene expression RPKM values for each experimental condition. Clustering was fitted to 20 distinct clustering groups, and the individual gene expression profiles clustered were further tested for enrichment of gene ontology GO terms associated with individual genes. Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . .", "Gene annotations were obtained from Reactome, a database of biological pathway and gene functional annotations . . Enrichment analysis was performed using two approaches. First, a hypergeometric test on GO annotations was carried out using an implementation of the GOStats package on each of the individual clusters obtained from k-means clustering . . In addition, gene set enrichment analysis GSEA was carried out on the entire filtered data set using 100,000 permutations, while duplicates were removed and an analysis of variance was applied. A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering.", "A total of 1,419 categories passed a minimum feature size of 10 and were used for further investigation. Cohorts of genes with shared patterns of expression over time were identified by k-means clustering. Those found to be enriched for DEGs were subsequently subjected to pathway analysis using the GeneMania system . . Using an ad hoc manual approach, relevant pathways and the connections between them were identified on the basis of existing data in the literature coupled with the temporal gene expression data obtained from this study. qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions.", "qRT-PCR analysis. Purified mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit Life Technologies according to the manufacturer's instructions. Analysis of the viral copy numbers was performed by quantitative reverse transcription-PCR qRT-PCR as previously described . . Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization.", "Host expression of the following genes was assayed with TaqMan assays indicated in parentheses : activating transcription factor 3 ATF3; Hs00231069_m1 , ATF4 Hs00909569_g1 , CEBPB Hs00270923_s1 , CEBPD Hs00270931_s1 , DDIT3 Hs00358796_g1 , FOS Hs04194186_s1 , JUN Hs01103582_s1 , EGR1 Hs00152928_m1 , IFI6 Hs00242571_m1 , IFIT1 Hs01911452_s1 , IFIT2 Hs01922738_s1 , IFIT3 Hs01922738_s1 , ISG15 Hs01921425_s1 , ISG20 Hs00158122_m1 , OASL Hs00984387_m1 , BIRC5 Mm00599749_m1 , and XIAP Mm01311594_mH . Assays for 18S rRNA Hs99999901_s1 or Mm04277571_s1 were used for normalization. Assays were performed according to the manufacturer's instructions using an ABI StepOne Plus instrument. Treatment with PERKi and collection for Western blot analysis. U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ .", "U87MG cells were pretreated for 2 h with 10 M the protein kinase RNAlike endoplasmic reticulum ER kinase PERK inhibitor PERKi GSK2606414 catalog number 516535; EMD Millipore or dimethyl sulfoxide DMSO in DMEM prior to infection with VEEV TrD MOI, 5 . After 1 h, the viral inoculum was removed and cells were washed with sterile PBS 1ϫ . The medium was replaced with medium containing the inhibitor or DMSO. At 16 hpi, the medium was removed, and the cells were washed with PBS and then collected for Western blot analysis. Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis.", "Knockdown of EGR1 with siRNA. U87MG cells seeded at 6.7 ϫ 10 4 cells per well in a 12-well plate were transfected with 50 nM siGenome Protein lysate preparation and Western blot analysis. Protein lysate preparation and Western blot analysis were performed as previously described . . Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis.", "Primary antibodies to the following were used: EGR1 antibody 44D5; catalog number 4154; Cell Signaling , polyclonal anti-Venezuelan equine encephalitis virus TC83 subtype IA/B capsid protein BEI Resources , CHOP antibody L63F7; catalog number 2895; Cell Signaling , phosphorylated ␣ subunit of eukaryotic initiation factor 2 p-eIF2␣; Ser51; antibody D9G8; catalog number 3398; Cell Signaling , ATF4 antibody D4B8; catalog number 11815; Cell Signaling , activated caspase 3 antibody Asp175; catalog number 9661; Cell Signaling , and horseradish peroxidase-conjugated ␤-actin catalog number ab49900-100; Abcam . Immunofluorescence analysis. U87MG cells were grown on coverslips in a 6-well plate, infected with VEEV TrD as described above, washed with PBS without Ca and Mg , and then fixed with 4% formaldehyde. Cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and then washed twice with PBS. The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100.", "The cells were blocked for 10 min at room temperature in 3% bovine serum albumin in PBS. Primary antibodies consisting of a VEEV capsid protein catalog number NR-9403; BEI Resources diluted 1:600 and an EGR1 antibody antibody 44D5; catalog number 4154; Cell Signaling diluted 1:400 were incubated in fresh blocking buffer at 37°C for 1 h and washed 3 times for 3 min each time in 300 mM NaCl with 0.1% Triton X-100. Alexa Fluor 568 donkey anti-goat secondary antibody catalog number A11057; Invitrogen and Alexa Fluor 488 donkey anti-mouse secondary antibody catalog number A21202; Invitrogen diluted 1:400 were used as secondary antibodies and treated in the same manner as the primary antibodies. DAPI 4=,6-di- amidino-2-phenylindole diluted 1:1,000 was used to visualize the nuclei. Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy.", "Coverslips were mounted onto glass slides using 10 l of Fluoromount G mounting medium catalog number 0100-01; Southern Biotech . A Nikon Eclipse TE2000-U fluorescence microscope was used for fluorescence microscopy. Images were viewed using a 60ϫ objective oil immersion lens. Five images of each sample were obtained, and a representative image of each sample is shown below. All images were subjected to fourline averaging. The images were processed through Nikon NIS-Elements AR Analysis v3.2 software. CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced.", "CellTiter Glo and Caspase 3/7 Glo assays. Wild-type and EGR1 Ϫ/Ϫ mouse embryonic fibroblasts MEFs were infected with TrD at various MOIs for an hour and then washed with PBS, and the medium was replaced. Cell viability was measured at 24 h postinfection using a Promega CellTiter luminescent cell viability assay catalog number G7571 according to the manufacturer's protocol. Luminescence was read using a Beckman Coulter DTX 880 multimode detector with an integration time of 100 ms per well. Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well.", "Similarly, caspase activation in infected wildtype and EGR1 Ϫ/Ϫ MEFs was measured at 24 h postinfection using a Promega Caspase 3/7 Glo assay catalog number G8090 according to the manufacturer's protocol. Luminescence was read using the DTX 880 multimode detector with an integration time of 100 ms per well. Nucleotide sequence accession numbers. The raw sequencing data for all RNA-Seq runs included in this work are publically available in the NCBI BioProject database under accession number PRJNA300864 http: //.nlm.nih.gov/bioproject/PRJNA300864 . VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . .", "VEEV replication kinetics in U87MG astrocytes. VEEV replicates in vivo in monocytes, macrophages, neurons, and astrocytes . . Common cell lines used to study VEEV infection include Vero and BHK cells; in this study, U87MG astrocytes were chosen as an in vitro model due to their physiological relevance and greater clinical significance. Initial experiments were performed to characterize viral replication in U87MG cells. VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 .", "VEEV replication kinetics in U87MG cells were measured using plaque assays and by monitoring viral protein and RNA expression levels and the cytopathic effect CPE on the infected cells Fig. 1 . Viral release was observed as early as 4 hpi, with ϳ4 log units of virus being observed, followed by a consistent increase in replication at 8 and 16 hpi Fig. 1A . Viral replication peaked at 16 hpi, and no additional increase in viral titers was observed at 24 hpi. Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B .", "Viral capsid expression followed a similar pattern, with protein being detected at 8 hpi and expression plateauing at 16 hpi Fig. 1B . Among infected U87MG cells, a significant CPE was observed by microscopy at 24 hpi, with little to no CPE being detected at 16 hpi data not shown . Consistent with these observations, increased caspase 3/7 activity was observed only at 24 hpi Fig. 1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile.", "1C . On the basis of these data, times of 4, 8, and 16 hpi, reflecting the early, middle, and late stages of the viral life cycle, respectively, were selected for RNA-Seq analysis in order to provide a dynamic view of the host-pathogen transcriptome profile. RNA sequencing analysis of VEEV-infected astrocytes. mRNA from triplicate sets of mock-and VEEV-infected U87MG cell cultures was isolated, purified at 4, 8, and 16 hpi, and used to prepare cDNA libraries for downstream RNA-Seq see Materials and Methods . A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig.", "A high-level summary of the RNA-Seq results is shown in Table 1 . VEEV RNA samples were assayed by quantitative RT-PCR at each time point as a control to demonstrate the increasing viral RNA load over time Fig. 1D , consistent with the increasing number of RNA-Seq reads mapped to the VEEV genome at later time points Table 1 . For RNA-Seq analysis, individual genes were expressed as the number of reads per kilobase of the exon model per million mapped reads RPKM . . Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material.", ". Log 2 -normalized RPKM expression values for each experimental sample are shown in Fig. 2A and can be found in Data Set S1 in the supplemental material. Minimal sample-to-sample variation in expression values within biological replicates was consistently detected R 2 Ͼ 0.89 for all replicates; data not shown . In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig.", "In addition, intersample variation was also found to be minimal when it was tested pairwise across the entire experiment by using RPKM values for ERCC97 synthetic spike-in control RNAs R 2 Ͼ 0.90 for all comparisons; data not shown . As anticipated, two-component principal component analysis of the RNA-Seq data for mock-infected cells versus VEEV-infected cells showed a clear separation of the samples at 16 hpi from the samples at earlier time points Fig. 2B . However, the clustering of VEEV-infected samples with mock-infected samples at earlier time points suggested that the response to viral infection was limited to a narrow subset of early response genes, thus placing a higher burden of proof on identifying differentially expressed genes DEGs during the first few hours of infection. Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al.", "Along these lines, two orthogonal methods were used to identify DEGs suitable for further characterization: the edgeR method . and the method developed by Baggerly et al. . . Genes identified by one method were provisionally considered DEGs, and those identified by both methods were candidate DEGs to be confirmed by qRT-PCR. In addition to comparing individual gene expression values for mock-infected cells and VEEV-infected cells at each time point, gene expression values were also compared serially within each time series of VEEV-infected cells for genes that did not show any statistically significant changes in expression in mock-infected cells. A schematic of the comparative analysis is shown in Fig. 2C .", "A schematic of the comparative analysis is shown in Fig. 2C . The number of statistically significant DEGs identified by each of these comparisons is shown in Fig. 2D . Furthermore, k-means clustering against normalized RPKM values was employed to identify gross changes in gene expression over time for cohorts of genes potentially sharing the same pathway or regulatory triggers Fig. 3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster.", "3 ; see also Data Set S2 in the supplemental material . Gene set enrichment analysis GSEA; see Material and Methods and Data Set S3 in the supplemental material was carried out on each kmeans cluster. In particular, cluster 20 Table 2 was significantly enriched for genes involved in translational control, the type I interferon-mediated signaling pathway, and the unfolded protein response UPR pathway GSEA P value Ͻ 0.01 . Although there is a well-established connection between translational control and UPR, a novel connection between UPR and the type I interferonmediated response in response to viral replication was suggested by pathway analysis see Materials and Methods , implicating early growth response 1 EGR1 as a potential bridge between these two pathways Fig. 4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3.", "4 . EGR1 belongs to cluster 20 and is strongly induced during VEEV infection, and several other genes associated with the interferon response belong to the same cluster: IRF1, IFIT1, IFIT2, ISG15, and ILF3. EGR1 has been associated with increases in the expression of activating transcription factor 3 ATF3 . , which is a key component of the UPR and which also belongs to cluster 20. This connection represented a potential a Biological process annotations obtained from Reactome for cluster 20. Reactome annotation identifiers are indicated for each annotation. Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle.", "Only traceable author submission TAS -classified annotations are considered. TAP, transporter associated with antigen processing; SRP, signal recognition particle. b Full set, the total number of genes in the genome with an annotated biological process; subset, total number of differentially expressed genes with an annotated biological process. Network of type I interferon response-and UPR-related genes. Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed.", "Large circles, differentially expressed genes; small circles, genes with no significant change in expression; red circles, type I interferon response factors; yellow circles, genes regulating DNA transcription; blue circles, unfolded protein response genes; red lines, genes involved in physical protein-protein interactions; blue lines, genes involved in a common pathway. This network was seeded with k-means clusters 18 and 20, and many ribosomal protein genes were removed. bridge between the UPR pathway and the interferon response pathway, with EGR1 being one of the potential key transcription factors driving this connection. Consequently, 15 genes from this analysis were selected for further characterization by qRT-PCR see below : ATF3, activating transcription factor 4 ATF4 , CEBPB, CEBPD, DDIT3/CHOP, EGR1, FOS, IFI6, IFIT1, IFIT2, IFIT3, ISG15, ISG20, JUN, and OASL. The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig.", "The expression values of these genes, as measured by RNA-Seq, are shown in Fig. 5A and B. Confirmatory qRT-PCR analysis indicated concordant gene expression Fig. 5C and D . The interferon response genes induced are in agreement with those detected in previously published studies . , and these genes served as an internal positive control. Moreover, the link between EGR1 and the interferon pathway has been demonstrated; EGR1 is induced by IFN-␥ in mouse fibroblasts and by IFN-␣, -␤, and -␥ in human fibroblasts . . EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated.", ". EGR1 and the UPR pathway were selected for further analysis, as their role in VEEV infection has not been elucidated. The RNA-Seq and pathway analysis data indicated that UPR and stress response genes were induced after VEEV infection. During an infection, host cells respond to cellular stresses resulting from increased viral protein translation and secretion by triggering the onset of the UPR pathway. The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 .", "The UPR pathway is an adaptive cellular response activated by endoplasmic reticulum ER stress due to protein misfolding. In order to regulate cellular homeostasis during protein folding and secretion, the UPR pathway has developed three classes of sensors to ensure proper cellular regulation: inositolrequiring enzyme 1 IRE1 , protein kinase RNA-like ER kinase PERK , and activating transcription factor 6 ATF6 . . During VEEV infection, the PERK arm of the UPR appeared to be altered, as two critical regulators of this pathway were differentially expressed: ATF4 and CHOP DDIT3 . . To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ .", ". To determine if DEGs altered subsequent protein expression, Western blot analysis was performed for CHOP, ATF4, and phosphorylated eIF2␣ p-eIF2␣ . Tunicamycin, a glycosylation inhibitor and inducer of UPR . , was included as a positive control. A time course analysis of U87MG cells treated with 1 M tunicamycin indicated that 8 h of treatment provided the most robust induction of UPR proteins data not shown . VEEV-infected but not mock-infected or UV-inactivated VEEV UV-VEEV -infected cells displayed a dramatic increase in p-eIF2␣ expression and a modest but consistent increase in CHOP and ATF4 expression at 16 hpi Fig. 6A . No change in protein expression was observed at 4 hpi data not shown .", "6A . No change in protein expression was observed at 4 hpi data not shown . Confocal microscopy confirmed CHOP and ATF4 up- regulation, demonstrating a more robust and nuclear staining pattern in VEEV-infected cells than in mock-infected cells Fig. 6C to E . While ATF4 protein expression levels increased, ATF4 mRNA abundances decreased following VEEV infection Fig. 5B and D . These results are consistent with the observation that ATF4 expression is regulated at the translational level upon UPR induction . . As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI .", ". As eIF2␣ can be phosphorylated by multiple kinases PERK, protein kinase double-stranded RNA dependent PKR , general control nonderepressible-2 GCN2 , and hemeregulated inhibitor HRI . , the PERK inhibitor PERKi GSK2606414 was used to determine if the observed phosphorylation was PERK dependent. Treatment of VEEV-infected cells with PERKi resulted in a marked decrease in eIF2␣ phosphorylation Fig. 6B . These results indicate that PERK contributes to eIF2␣ phosphorylation but that there is likely an additional kinase contributing to the phosphorylation event. Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus.", "Collectively, these findings indicate that the PERK arm of the UPR pathway is induced at later time points following VEEV infection. EGR1 is upregulated in infected cells and localizes to the nucleus. EGR1 is a transcription factor that can be induced by numerous signals, including oxidative stress, hypoxemia, and growth factors . . It can also be activated upon infection by both DNA and RNA viruses, including Epstein-Barr virus, mouse hepatitis virus, murine coronavirus, and Japanese encephalitis virus . . . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . .", ". . . Treatment of MEFs with the UPR activator thapsigargin has been shown to induce EGR1 expression in a PERK-dependent manner . . Given the link between EGR1 and UPR and the robust induction of EGR1 mRNA expression following VEEV infection Fig. 4 and 5 , EGR1 was chosen for further study. EGR1 protein expression after VEEV infection was analyzed by Western blot analysis. As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig.", "As previous studies have indicated that EGR1 can be activated by mouse hepatitis virus independently of virus replication likely due to cellular membrane disruption following entry ., a UV-inactivated virus control UV-VEEV was included. EGR1 protein levels were increased following VEEV infection compared to those in mock-infected cells and UV-VEEV-infected cells Fig. 7A; compare lanes 3, 6, and 9 . The most dramatic upregulation of EGR1 occurred at 16 hpi; this correlates with the highest levels of VEEV capsid production Fig. 1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . .", "1B . Following induction, EGR1 has been shown to translocate to the nucleus to induce gene expression through binding to the Egr binding sequence EBS GCG G/T GGCG . . Confocal microcopy revealed high levels of EGR1 in the nuclei of infected cells, whereas only low levels of both nuclear and cytoplasmic EGR1 were detected in mock-infected cells Fig. 7B . PERKi treatment of VEEV-infected cells resulted in a complete loss of EGR1 induction Fig. 7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK.", "7C , indicating that EGR1 was induced in a PERK-dependent fashion. These results demonstrate that EGR1 protein levels and nuclear localization are increased following VEEV infection and that the induction of EGR1 is dependent on PERK. The loss of EGR1 inhibits VEEV-induced apoptosis but does not alter VEEV replication kinetics. As EGR1 influences cell survival and apoptosis . , the impact of EGR1 on VEEV-induced cell death was assessed. Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A .", "Caspase 3 cleavage was observed in WT MEFs at 24 hpi when they were infected at an MOI of 0.5 and started as early as 16 hpi when they were infected at an MOI of 5 Fig. 8A . In contrast, EGR1 Ϫ/Ϫ cells showed little to no detectable caspase cleavage following infection with VEEV. Two sets of experiments were performed to quantitatively confirm these results: CellTiter Glo assays to measure total cell viability ATP production and Caspase 3/7 Glo assays to measure caspase 3/7 activity. Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B .", "Both WT and EGR1 Ϫ/Ϫ MEFs displayed dose-dependent decreases in cell viability following VEEV infection, with EGR1 Ϫ/Ϫ cells having significantly more viable cells at each MOI examined Fig. 8B . Concordantly, a dose-dependent increase in caspase 3/7 activity was observed following VEEV infection, with EGR1 Ϫ/Ϫ cells demonstrating reduced caspase 3 activity at MOIs of 0.5 and 5 Fig. 8C . These results were replicated in U87MG cells transfected with siRNA targeting EGR1 Fig. 8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . .", "8D . EGR1 has been shown to negatively regulate the transcription of BIRC5 survivin , an inhibitor of apoptosis IAP family member . . RNA-Seq data indicated that BIRC5 gene expression was decreased following VEEV infection: log 2 -transformed fold change values of normalized gene expression were Ϫ1.16, Ϫ1.18, and Ϫ1.50 at 4, 8, and 16 hpi, respectively see Table S1 in the supplemental material and NCBI BioProject accession number PRJNA300864 . WT and EGR1 Ϫ/Ϫ MEFs were used to determine if EGR1 influenced BIRC5 gene expression following VEEV infection. BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E .", "BIRC5 expression was significantly decreased at 16 hpi in VEEV-infected WT MEFs, but this reduction was not observed in VEEV-infected EGR1 Ϫ/Ϫ MEFs Fig. 8E . Ex-pression of the gene for the X-linked inhibitor of apoptosis XIAP , another IAP family member, was not significantly differentially altered after infection data not shown . Collectively, these results demonstrate that EGR1 contributes to VEEV-induced apoptosis. VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay.", "VEEV replication kinetics were determined for both EGR1 Ϫ/Ϫ and WT MEFs to determine the relevance of EGR1 in viral replication. Cells were infected at two different MOIs 0.5 and 5 , and viral supernatants were collected at 4, 8, 16, and 24 hpi and analyzed by plaque assay. The replication kinetics were similar between EGR1 Ϫ/Ϫ and WT MEFs at both MOIs, with titers peaking at 16 hpi Fig. 9A . A lack of EGR1 expression was confirmed by Western blotting Fig. 9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig.", "9B . These results were replicated in U87MG cells transfected with siRNA targeting EGR1. Transfection of siRNA targeting EGR1 resulted in a Ͼ90% decrease in EGR1 protein expression Fig. 9D without any significant effect on viral replication Fig. 9C . These results suggest that the decrease in apoptosis observed in EGR1 Ϫ/Ϫ MEFs was not due to altered VEEV replication kinetics. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms of alphaviruses, largely due to a knowledge gap in the host-pathogen interactome. VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . .", "VEEV infection often results in fatal encephalitis and is known to inhibit both cellular transcription and translation in order to downregulate the innate immune response . . In contrast, in the CNS VEEV has been shown to upregulate numerous genes in both the inflammatory response and apoptotic pathways . . Specifically, numerous proinflammatory cytokines, including interleu-kin-1␤ IL-1␤ , IL-6, IL-12, glycogen synthase kinase 3␤, inducible nitric oxide synthase, and tumor necrosis factor alpha TNF-␣ , have all been shown to play a role in VEEV pathogenesis . . . . . .", ". . . . . The use of high-throughput next-generation sequencing technologies, such as RNA-Seq, allows an in-depth and unbiased look into the virus-host transcriptome, thus enabling changes in the expression of specific mRNAs to be connected with phenotypic outcomes. To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation.", "To this end, identification of critical differentially expressed transcripts among clinically relevant infected cells will help lead to a greater understanding of viral pathogenesis and may prove beneficial for the identification of therapeutic targets. In this study, network analysis/RNA-Seq data and the results of protein expression studies revealed that VEEV infection resulted in activation of the PERK arm of the UPR pathway, including the activation of ATF4, CHOP, and eIF2␣ phosphorylation. Several alphaviruses have previously been reported to hijack key components of the UPR pathway in order to promote viral replication, as the reliance of enveloped viruses on the ER for the synthesis of viral envelope-associated glycoproteins and their transport to the plasma membrane often stresses the ER due to rapid viral protein production . . Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication .", "Modulation of the UPR is not unique to alphaviruses; rather, it is a shared trait of many positive-sense RNA viruses. Dengue virus has been shown to suppress PERK by inhibiting continued eIF2␣ phosphorylation in order to inhibit immediate apoptosis, increasing viral protein translation and extending the length of productive viral replication . . Studies with hepatitis E virus HEV have demonstrated that expression of HEV capsid protein open reading frame 2 ORF2 activates the expression of CHOP and ATF4 . . In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . .", ". In HEV, ORF2 was shown to stimulate CHOP through both ER stressors and amino acid response elements AARE through interaction with ATF4 . . The results shown here indicate that during VEEV infection, initiation of the UPR pathway and subsequent activation of EGR1 play a role in the outcome of virus-induced apoptosis. During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1.", "During the initial detection of ER stress, PERK is able to identify misfolded proteins in the lumen of the ER and phosphorylates eIF2␣ in order to initiate prosurvival pathways in the UPR through the general At 24 hpi caspase 3/7 activity was analyzed using the Caspase 3/7 Glo assay. The fold change values for mock-infected cells were set to a value of 1. **, P Ͻ 0.001. E EGR1 Ϫ/Ϫ and WT MEFs were mock or VEEV infected MOI, 5 . RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method.", "RNA was prepared, and gene expression was determined by qRT-PCR using a TaqMan assays for BIRC5 survivin . The data shown are the values of the fold change of normalized gene expression determined by the ⌬⌬C T threshold cycle C T method. *, P Ͻ 0.005 comparison of VEEV-infected WT and EGR1 Ϫ/Ϫ cells . inhibition of protein synthesis . . VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4.", ". VEEV appears to induce the UPR and promote increased eIF2␣ phosphorylation, which results in the translational inhibition of most mRNAs, while UPR selectively increases the translation of ATF4. ATF4 is responsible for the expression of genes that encode proteins involved in apoptosis, redox processes, amino acid metabolism, and ER chaperone recruitment and is a well-known mediator of the PERK pathway and CHOP . . CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . .", ". CHOP activation facilitates the increased expression of cellular chaperones in order to counteract the buildup of misfolded proteins . . Failure to suppress protein misfolding in persistently stressed cells, such as during a viral infection, can then result in activation of the proapoptotic transcription factor CHOP, leading to suppression of the antiapoptotic protein B cell lymphoma-2 Bcl-2 . CHOP can also function as a prosurvival transcription factor by dephosphorylating eIF2␣ through activation of the DNA damage-inducible protein GADD34 in a self-regulating feedback look . . However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis.", ". However, the data presented here support a model whereby VEEV infection leads CHOP to function in its proapoptotic role, as no change in GADD34 gene expression was detected by RNA-Seq analysis. While the UPR was induced following VEEV infection, robust activation was not observed until later time points after infection. This is somewhat surprising, as VEEV infection is expected to induce significant ER stress due to the massive production of viral proteins during the course of an acute robust infection. The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones .", "The structural proteins of VEEV are translated from the viral subgenomic RNA into polyproteins on the rough ER. The E1 and pE2 precur-sor glycoproteins are then assembled as heterodimers in the ER, undergoing conformational changes requiring numerous chaperones . . It is possible that VEEV has developed mechanisms to subvert the induction of the UPR. In order to counteract the UPR, the nonstructural proteins nsPs of Chikungunya virus CHIKV have been shown to inhibit expression of ATF4 and other known UPR target genes, including GRP78/BiP, GRP94, and CHOP . . Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation.", ". Through nsP activity, CHIKV has developed a means of suppressing the UPR activity resulting from viral glycoprotein-induced ER stress, thus preventing immediate autophagy and apoptotic activation. The VEEV capsid is responsible for interfering with nucleocytoplasmic trafficking and inhibiting rRNA and mRNA transcription and has been implicated in the regulation of type I IFN signaling and the antiviral response through the regulation of both viral RNA and protein production . . Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR.", "Therefore, we hypothesize that the ability of the VEEV capsid to inhibit cellular transcription and block nucleocytoplasmic trafficking results in delayed induction of the UPR. The results of a detailed network analysis based on existing data in the literature, coupled with the temporal gene expression profiles obtained from this study, point toward EGR1 being an important node in the novel link between VEEV activation of the type I interferon response and UPR. EGR1 is known to form a DNA binding complex with C/EBPB, a critical dimerization partner of CHOP . . Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . .", "Previous studies have demonstrated that the nuclear localization of CHOP may act as an inducer of EGR1 and that CHOP may act as a transcriptional cofactor for regulation of C/EBPB-EGR1 target genes . . The results of the Western blot and microscopy analysis presented in this study support this model, as VEEV infection was found to increase both the overall levels and the nuclear distribution of CHOP along with those of EGR1. Previous studies demonstrated EGR1 mRNA induction by IFN-␥ in mouse fibroblasts and by TNF-␣, TNF-␤, IL-1, IFN-␣, IFN-␤, and IFN-␥ in human fibroblasts . . EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor.", ". EGR1, also known as Zif268 and NGF1-A, is a zinc finger protein and mammalian transcription factor. It has been implicated in cellular proliferation and differentiation, but it may also have proapoptotic functions, depending on the cell type and stimulus . . Of particular interest, EGR1 directly controls proliferation when activated by the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway in mitogen-stimulated astrocytes . . Virus-induced changes in EGR1 expression have been observed in several in vitro systems. In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . .", "In HIV-1-infected astrocytes, EGR1 upregulation was found to be induced by Tat through transactivation of the EGR1 promoter, leading to cellular dysfunction and Tat-induced neurotoxicity . . Increased amounts of EGR1 mRNA have also been demonstrated to act in a region-specific manner, corresponding temporally with viral RNA production in the brain tissues of rats infected with either rabies virus or Borna disease virus . . In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection.", "In summary, the current study demonstrates a potential link between UPR activation and EGR1. EGR1 Ϫ/Ϫ MEFs demonstrated lower levels of susceptibility to VEEV-induced cell death than wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following infection. Studies are under way to determine if alteration of the UPR through small molecule inhibitors or siRNA interference influences VEEV replication and/or cell death. To date the mechanisms underlying VEEV pathogenesis and subsequent neuronal degeneration have been only partially elucidated. Therefore, determining the role of EGR1 and UPR may play a significant role in the development of a novel therapeutic target resulting in decreased neuronal death and the subsequent neuronal sequelae that result from infection." ]

[ "Bacterial and viral co-infections of the respiratory tract are life-threatening and present a global burden to the global community. Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes are frequent colonizers of the upper respiratory tract. Imbalances through acquisition of seasonal viruses, e.g., Influenza A virus, can lead to bacterial dissemination to the lower respiratory tract, which in turn can result in severe pneumonia. In this review, we summarize the current knowledge about bacterial and viral co-infections of the respiratory tract and focus on potential experimental models suitable for mimicking this disease. Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available.", "Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available. The knowledge gained from these studies led to important discoveries and advances in understanding these infectious diseases. Nevertheless, mouse and other infection models have limitations, especially in translation of the discoveries to humans. Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 .", "Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 . The upper respiratory tract is a natural niche for potentially pathogenic bacteria embedded in commensal communities forming the nasopharyngeal microbiome. In particular, the microbial communities of the nasopharynx . are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . .", "are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . . The composition of the nasopharyngeal microbiome is highly dynamic Biesbroek et al., 2014a,b,c and many factors, including environmental and host factors, can affect microbial colonization . . Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b .", "Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b . The dynamic microbiome composition is guaranteed through the interplay between bacterial species, other microbes, and changing environmental conditions, as well as host-bacteria interactions Blaser and Falkow, 2009 . Most of the time, the microbiome and its interplay with the human host are believed to be beneficial for both Pettigrew et al., 2008; Murphy et al., 2009 . However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues .", "However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues . . While commensal respiratory microbiota facilitated immune-support against Influenza A virus infection IAV , oral treatment with antibiotics resulted not only in a shift of bacterial composition, but also in impaired CD4 T-, CD8 T-, and B-cell immunity following infection with IAV in mice . . Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls .", ". Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls . . However, another study published in the same year contradicted these results . . Chaban and colleagues analyzed microbiomes of 65 patients from H1N1 IAV outbreak in 2009. Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . .", "Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . . In this review we discuss secondary bacterial infections of the respiratory tract after primary infection by IAV with a focus on mechanisms by which these interactions are potentially mediated, and we will provide insight into the host contribution and immunological consequences. We further focus on potential animal models suitable for mimicking asymptomatic bacterial colonization and disease progression and thus, enabling to study adaptation strategies, viral-bacterial interactions, and immune responses in these highly lethal co-infections. Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . .", "Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . . The 8-segmented genomes of influenza A viruses are characterized by a significant plasticity. Due to point mutations and re-assortment events new variants or strains with epidemic or pandemic potential emerge Neumann et al., 2009 . In addition, influenza can be transmitted between animals, including swine, birds, horses, and humans, making it a zoonotic disease . . Seasonal influenza usually resolves without consequences in healthy individuals.", ". Seasonal influenza usually resolves without consequences in healthy individuals. However, it is estimated that seasonal influenza effects 5-10% of the world's population resulting in about 250,000 to 500,000 deaths annually Tjon-Kon-Fat et al., 2016 . At greater risk to develop secondary bacterial pneumonia are individuals with comorbidities, elderly people age > 65 , pregnant women, and children under the age of one . . For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 .", ". For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 . The pandemic killed around 50 million people worldwide and remains unique in its severity compared to other big outbreaks. However, many of the findings have been reinterpreted in recent years Brundage and Shanks, 2007; Chien et al., 2009 . It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . .", "It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . . Individual studies limited to certain regions identified also other pathogens commonly colonizing the respiratory tract, including Staphylococcus aureus, group A streptococcus GAS and Haemophilus influenzae Brundage and Shanks, 2008 . During the next two pandemics H2N2 Asian Flu 1957 and H3N2 Hong Kong Flu 1968 −1969 bacterial co-infections were less likely the cause of death compared to the Spanish Flu Giles and Shuttleworth, 1957; Trotter et al., 1959 . Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 .", "Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 . In 1957-1958, S. aureus was predominantly isolated from fatal pneumonia cases Hers et al., 1957 Hers et al., , 1958 Robertson et al., 1958; Martin et al., 1959 , whereas S. pneumoniae returned as predominant cause of severe pneumonia during the Hong Kong Flu Sharrar, 1969; Bisno et al., 1971; Burk et al., 1971; Schwarzmann et al., 1971 . Forty years later in 2009, a novel H1N1 virus of swine origin emerged and caused again a pandemic Dawood et al., 2009 Dawood et al., , 2012 . In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 .", "In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 . Although regional variations occurred, pneumococci and S. aureus were the most frequently isolated bacterial species Mauad et al., 2010; Shieh et al., 2010; Rice et al., 2012 . Group A streptococcus was absent in many local pneumonia outbreaks associated with viruses, but was predominant in others Brundage and Shanks, 2008; Ampofo et al., 2010 . When it does appear, it is typically third in incidence . .", "When it does appear, it is typically third in incidence . . Overall, data on pandemic outbreaks suggest that disease severity and mortality can be linked to secondary bacterial pathogens with variations depending on regions and state of immunity of the population Brundage and Shanks, 2008; Shanks et al., 2010 Shanks et al., , 2011 McCullers, 2013 . There is increasing evidence that the nasopharyngeal microbiota plays an important role in the pathogenesis of acute viral respiratory infections Teo et al., 2015; de Steenhuijsen Piters et al., 2016; Rosas-Salazar et al., 2016a,b . Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens.", "Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens. All of them are frequent colonizers of the human nasopharynx and they share many features including pathogenic mechanisms and clinical aspects Figure 1 . However, they also have unique properties. Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia.", "Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia. In the last decades, the pathogen became resistant to an increasing number of antibiotics and methicillin-resistant S. aureus MRSA is now a major cause of hospital acquired infections Hartman and Tomasz, 1984; Ubukata et al., 1989; Zetola et al., 2005 . Also the rise of community-acquired S. aureus strains is of special concern, because certain clones are associated with very severe infections . . Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 .", ". Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 . The pneumococcus is a typical colonizer of the human nasopharynx. About 20-50% of healthy children and 8-30% of healthy adults are asymptomatically colonized McCullers, 2006 . Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 .", "Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 . Group A streptococci colonize the mouth and upper respiratory tract in about 2-5% of world's population Okumura and Nizet, 2014 . The most common, non-invasive and mild infections caused by GAS are tonsillitis and pharyngitis with estimated 600 million cases per year . . Listed as number nine in the list of global killers with around 500,000 deaths annually .", ". Listed as number nine in the list of global killers with around 500,000 deaths annually . , it is obvious that this pathogen can cause severe invasive infections, including pneumonia, sepsis, streptococcal toxic shock syndrome, and necrotizing skin infections Cunningham, 2000; Carapetis et al., 2005 . Although all three pathogens are able to cause highly lethal diseases, the most fatal remains the pneumococcus, estimated to cause ca. 10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . .", "10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . . Influenza A virus binds via HA to either α2,3or α2,6-linked sialic acid at the surface of epithelial cells of the upper and lower respiratory tract . . Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways.", "Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways. Binding of viral RNA to retinoic acid inducible gene 1 induces the expression of type I and III interferons and activates transcription factor NF-κB, which in turn activates the release of pro-inflammatory cytokines Durbin et al., 2013; Iwasaki and Pillai, 2014 . In addition, inflammasome activation leads to the release of IL-1β and IL-18 Pothlichet et al., 2013; Iwasaki and Pillai, 2014 . All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 .", "All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 . Furthermore, aberrant coagulation induced by virus infection causes a hyper-inflammatory response Yang and Tang, 2016 . All these events contribute to lung tissue injury Imai et al., 2008; Davidson et al., 2014 . The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . .", "The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . . On the other hand, already colonized bacteria might enhance influenza virus virulence either by directly secreting proteases that cleave and activate HA Figure 2 Bottcher-Friebertshauser et al., 2013 or, indirectly, by activating host proteases such as plasminogen, which increases replication rates and infectivity of the virus Scheiblauer et al., 1992; Tse and Whittaker, 2015 . Potentially pathogenic bacteria, including the three species mentioned above, express an arsenal of virulence factors responsible for attachment to human host structures. Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract.", "Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract. Seasonal IAV infection can lead to an increased risk of secondary bacterial infections, i.e., pneumonia. Several experimental models can be used for studying these severe infections. Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases.", "Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases. In vivo bacterial and viral co-infections are mainly performed in mice, which does not necessarily resemble the human physiology and immune system. Thus, we suggest using the porcine model, which nearly resembles over 80% of the human immune system. MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 .", "MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 . Once the initial attachment occurs, bacterial cytotoxins including pneumolysin of pneumococci Garcia-Suarez Mdel et al., 2007; Zahlten et al., 2015 , α-hemolysin and leukocidins of S. aureus . , and Streptolysins S and O and Streptococcal pyrogenic exotoxin B of S. pyogenes Tsai et al., 1998; Gurel et al., 2013; Siemens et al., 2015 Siemens et al., , 2016 , can synergize with viral counterparts to further increase lung tissue pathology. Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 .", "Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 . As the patient begins to recover from viral infection, secondary bacterial infections might occur . due to the incomplete wound healing and exposure of host membrane components, including laminin, collagens type I and IV to classical bacterial MSCRAMMs Louria et al., 1959; Puchelle et al., 2006 . Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 .", "Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 . Respiratory viruses like IAV are able to induce suppression and killing of the resident alveolar macrophages Figure 2 . . These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections .", "These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections . . In addition, induction of interferons as a response to viral infection compromises the immune sensing of Gram-positive bacteria by neutrophils and macrophages, which would normally clear the bacteria from the lungs Figure 2 Sun and Metzger, 2008; Tian et al., 2012 . The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state .", "The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state . . The subsequent infection with Gram-positive bacteria, e.g., pneumococci, enhances the type I interferon expression, which in turn suppresses production of the CCL2 chemokine and recruitment of macrophages Nakamura Frontiers in Microbiology | FIGURE 2 | The interplay between IAV, bacteria, and the human host. The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment.", "The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment. IAV is able to induce suppression and killing of resident alveolar macrophages AM , which in turn delays viral clearance. The release of viral RNA activates different immune response pathways resulting in cytokine storm. Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages.", "Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages. After initial inflammation, the situation might worsen due to cellular infiltration of the lungs by neutrophils PMN , leading to an increased degranulation and tissue damage by effector molecules, including heparin-binding protein HBP . et al., 2011 . Another study by Shahangian et al. . revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion.", ". revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion. Other studies found that IAV exposed lungs had impaired natural killer NK cell responses in the airway to subsequent S. aureus infection . . Reduced TNFα production by NK cells was identified as a crucial upstream mechanism of depressed antimicrobial activities by alveolar macrophages Figure 2 . . It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . .", "It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . . In vitro studies on the interplay between IAV-pneumococci and human dendritic cells revealed TLR3 as a crucial sensor of viral and bacterial RNA leading to enhanced IL-12p70 production, which in turn might promote an anti-viral state by upregulation of interferons Yamamoto et al., 2004; Spelmink et al., 2016 . However, it should be noted that depending on the bacterial species the disease manifestation and underlying innate immune responses might vary Sharma-Chawla et al., 2016 . A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 .", "A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 . These processes are characterized by a general anti-inflammatory state and suppression of mechanisms involved in pathogen clearance due to increased interleukin-10 production van der Sluijs et al., 2004; Metzger and Sun, 2013 . The anti-inflammatory state suppresses the expression of pattern recognition receptors PRR on professional phagocytes leading to impaired phagocytosis and killing of microbes. These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis .", "These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis . . In co-infections, after initial inflammation in response to viral infection the situation might worsen due to bacterial invasion and enhanced cellular infiltration of the lungs by neutrophils, leading to an increased tissue damage and cytokine storm Figure 2 Conenello et al., 2007; McAuley et al., 2007 McAuley et al., , 2010 Porto and Stein, 2016 . Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 .", "Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 . Bacterial pathogens also express a variety of cytolytic toxins that can contribute to inflammation and tissue pathology. Pneumolysin, a pneumococcal pore-forming toxin with low affinity to lung epithelial cells, can damage neutrophils by utilizing P2X7 receptor . . Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs .", ". Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs . . GAS toxins, including SLO and SpeB, are capable of directly causing tissue damage and promoting pro-inflammatory states through neutrophil lysis Snall et al., 2016; Uhlmann et al., 2016 . The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 .", "The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 . Figure 2 summarizes the interplay between virus, bacteria, and host. Experimental animal models are a useful tool to study in vivo effects of different infectious agents and they represent approximately 3% of all pneumonia research published in peerreview journals . . However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 .", ". However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 . Hackam and colleagues identified 2,000 articles published between 1980 and 2006 in seven leading scientific journals that regularly publish animal studies Hackam and Redelmeier, 2006 . Seventy-six out of 2,000 were highly cited with a median citation count of 889. Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 .", "Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 . In pneumonia models, mammalians are mostly used because of their anatomical and physiological proximity to humans . . To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms.", "To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms. Rapid reproductive rate, small size, less complicated handling, the ability to reproduce and compare results with already published bacterial and viral mono-infections, detailed knowledge of genetics and immune responses, and a plethora of available reagents to study infections in mice are reasons for the use of these animals. To avoid variations in responses due to genetic diversity inbred mice strains are useful tools for studies aiming to elucidate molecular mechanisms of diseases. In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 .", "In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 . However, mouse models for bacterial and/or viral infections have several limitations. Most of the bacterial and viral species under study are human pathogens. In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 .", "In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 . IAV was shown to be essential for pneumococcal transmission from colonized mice to their naive littermates and the transmission occurred only when all mice were infected with IAV . . et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 .", ". et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 . The infection of ferrets with human seasonal IAV isolates results in an upper respiratory tract infection similar to human influenza infection Tripp and Tompkins, 2009 . In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism.", "In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism. A report by Sanford and Ramsay showed enhanced staphylococcal colonization of the upper respiratory tract in IAV infected animals as compared to non-infected, while no difference between both groups was observed in group B streptococcal infection Sanford and Ramsay, 1987 . In contrast, Smith and Mc Cullers reported lack of establishment of staphylococcal infection even when ferrets were pre-infected with IAV Smith and McCullers, 2014 . The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 .", "The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 . In recent years, the guinea pig Cavia porcellus was also used in pneumonia research. The physiology and anatomy of the guinea pig lung resembles to a certain extent the human lung and this model organism is often used in non-infectious lung diseases, including asthma and chronic obstructive pulmonary disease Canning and Chou, 2008 . In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses.", "In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses. Although viral replication can be readily detected upon intranasal inoculation in the upper respiratory tract and the lungs, guinea pigs exhibit only minor clinical symptoms Lowen et al., 2006; Gabbard et al., 2014 . However, the lung pathology of human IAV infected guinea pigs correlates with the clinical severity of human infection . . Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . .", ". Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . . Guinea pigs infected with pneumococci alone and cage-mated with non-treated contact animals transmitted the bacteria only in 7% of cases, while Sendai-virus infected, co-housed guinea pigs acquired pneumococcal infection in 83% of contacts . . Another study evaluated antibiotic efficacy in invasive pulmonary infection caused by penicillin resistant pneumococcus . . Intratracheal instillation of 3 × 10 9 CFU of S. pneumoniae induced a fatal pneumonia and bacteremia in 85% of untreated animals within 46 h . . As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents.", ". As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents. This is in parts due to the lack of species specific reagents, which is a disadvantage in using this model organism. Recently, the cotton rat Sigmodon hispidus was reported to be susceptible to IAV. Nasal and pulmonary infection in adult inbred cotton rats did not require viral adaptation . . The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days.", "The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days. Tissue pathology included damage of bronchiolar epithelium and the animals developed pneumonia which persisted for nearly 3 weeks . . In bacteriological studies rats are more frequently used. There are numerous rat models investigating the impact of diabetes . , metabolic syndromes . , cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors .", ", cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors . on development of pneumococcal, streptococcal, and staphylococcal pneumonia and lung pathology. Unfortunately, there are only few studies on bacterial and viral co-infections in rats. The first was performed by Harford et al., 1946 Harford et al., 1946 . The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . .", "The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . . Another study on human respiratory syncytial virus and S. pneumoniae revealed that rats were easily colonized with pneumococci, but viral replication after subsequent infection was strain dependent. In addition, neither pneumococci nor the virus spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates . Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents.", "Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents. Rabbits Oryctolagus cuniculus are well known for their use in studying cardiovascular diseases, antibody production, and eye research. Rabbits were also employed to study pneumonia, although only a few models are available. Typical read-out parameters include survival, leukocyte infiltration of the lungs, lung pathology, and assessment of drug concentration in serum. One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . .", "One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . . This study revealed that rabbits possess an active immunity if they have recovered from one attack of experimental pneumonia and they may subsequently resist repeated intra-tracheal dosages of pneumococci Kline and Winternitz, 1913 . In 1926 an infection by inhalation of Type I pneumococci was established in rabbits Stillman and Branch, 1926 . The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 .", "The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 . However, this infection route requires surgery and species-specific reagents are scarce. In IAV research rabbits are frequently used for antibody production and for studies on antibody kinetics following single or multiple IAV administrations Loza-Tulimowska et al., 1977 . Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp.", "Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp. was investigated revealing that nasally and orally inoculated cottontails shed relatively large quantities of viral RNA . . Notably, low viral titers were found to be sufficient to initiate viral replication in cottontails . . However, despite their susceptibility to IAV infection, rabbits are only rarely used as model for IAV pathogenesis since they offer no improvement over other established infection models. Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable.", "Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable. The species most commonly used are rhesus macaques Macaca mulatta and cynomolgus macaques Macaca fasciluraris . Although it was shown early that macaques were susceptible to IAV . , the animal models of choice remained ferrets and mice. Recently, macaques have been used to compare the pathogenesis of highly virulent 1918 pandemic IAV and the pathogenic bird flu strain H5N1 with a conventional H1N1 strain . . Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . .", ". Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . . The 1918 IAV strain induced dysregulation of the antiviral response leading to insufficient protection of the host, which in turn resulted in acute respiratory distress and a fatal outcome . . The 2009 pandemic H1N1 US isolate caused severe pathological lesions in the lungs of the macaques Itoh et al., 2009 . The three studies mentioned above used combined intratracheal delivery of high doses of virus. A recent study by Marriott et al. analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . .", "analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . . Virus replication was detected in all challenge groups, although the disease remained sub-clinical. In bacteriological studies non-human primates are rarely used. For group A streptococcal infection longitudinal transcriptome analyses were performed in experimental pharyngitis . and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a .", "and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a . Another study by Olsen and colleagues analyzed the contribution of PVL of a highly virulent USA300 S. aureus strain in respiratory infection Olsen et al., 2010b . Although the lower respiratory tract disease observed in monkey mimicked the clinical and pathological features of early mild to moderate pneumonia in humans, no involvement of PVL in lung pathology or immune cell influx of the lungs could be detected Olsen et al., 2010b . The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . .", "The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . . Pneumonia progression was monitored by clinical parameters assessment, blood chemistry, nasal swabs, and pathology of the lungs. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but did not predispose the animals to subsequent severe infection with the USA300 clone . . Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade.", "Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade. In 2013, Philipp and colleagues analyzed the carriage rate of pneumococcus in 158 colony animals. None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci.", "None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci. But, when infants were colonized with 19F strain via nasopharyngeal instillation, the colonization was induced in eight of eight infants, lasted for 2 weeks in all animals and for 7 weeks in more than 60% . . The same group tested detoxified pneumolysin dPly and pneumococcal histidine triad protein D PhtD as potential vaccine candidates to prevent pneumonia . . After immunization the rhesus macaques were challenged with a 19F pneumococcal strain.", ". After immunization the rhesus macaques were challenged with a 19F pneumococcal strain. AS02-adjuvanted PhtD-dPly vaccine protected the animals against S. pneumoniae-induced pneumonia, which was linked to the capacity i to greatly reduce bacterial load within the first week post-challenge and ii the levels of PhtD-and Ply-specific antibodies . . Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified.", "Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified. Small mammals including rodents are well known from a biological, genetic, and immunological point of view and are easy to maintain. The choice of these particular animals for infectious disease studies is often a result of a compromise between technical and financial options. However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans.", "However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans. Primates are usually legally reserved to specific topics. In this case, pigs could be an appropriate model system for studying infectious diseases including pneumonia Figure 1 . The composition and size of the porcine genome is comparable to that of humans . . In addition, human and porcine organs have many common features and functions . . The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar.", ". The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar. Furthermore, like humans, pigs possess tonsils, which are absent in mice . . A major advantage of studying infectious diseases by utilizing pigs as a host organism is that pigs have a full set of innate and adaptive immune effectors. According to whole genome sequencing results the porcine immune system resembles over 80% of the human immune system, whereas mice share less than 10% with humans . . Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 .", ". Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 . In contrast to mice and similar to humans, pigs have 50-70% of circulating polymorph nuclear cells . . In addition, all functional cytokines or orthologs involved in Th1, Th2, Th17, and Treg paradigm and corresponding immune cells have been described in pigs Murtaugh et al., 2009; Kaser et al., 2011; Kiros et al., 2011 . Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . .", "Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . . In contrast to human monocytes, which can be divided in three subclasses classical CD14 + CD16 − , nonclassical CD14 + CD16 + , and intermediate CD14 ++ CD16 + , porcine monocytes consist of four subclasses Chamorro et al., 2005; Fairbairn et al., 2013 . Like human monocytes they express adhesion molecules, such as VLA-4 and LFA-1 and costimulatory molecules, including CD80 and CD86 . . The pig has previously been used to mimic a number of human infectious diseases.", ". The pig has previously been used to mimic a number of human infectious diseases. Examples for S. aureus infections with this model organism are wound infections Svedman et al., 1989 , osteomyelitis . , and sepsis . . Intravenous inoculation of piglets with pneumococci led to bacteremia during a 5 days period and was associated with fever and septic arthritis. Intranasal inoculation of piglets led to colonization for at least six consecutive days without causing clinical signs . . In addition, research on respiratory infections of pigs by human pathogens including S. aureus . , Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years.", ", Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years. The fact that pigs and humans are infected with identical subtypes of IAV H1N1, H3N2 , and show similar clinical presentation and pathogenesis, makes pigs an ideal model organism for studies on respiratory co-infections . . Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis.", "Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis. S. suis usually inhabits mucosal surfaces of tonsils, nares, genital and alimentary tract of piglets. Once the microbial balance is disturbed, the bacteria can cause meningitis, septicemia, arthritis, and pneumonia in pigs . . Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent.", ". Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent. In general, serotype 2 is most frequently isolated from diseased pigs . . S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 .", "S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 . A recent publication by Lin and colleagues on H1N1 and S. suis co-infected piglets demonstrated the synergistic effects of both pathogens . . Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . .", ". Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . . In addition, genes associated with immune responses, inflammatory cytokine production, and apoptotic pathways were highly overexpressed in the coinfected group . . Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals.", ". Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals. Although the use of animals contributes greatly to our understanding of infectious diseases, human 3D-organotypic tissue models and ex vivo organ tissues should be considered, as they are most valuable tools to study host-pathogen interactions in a more complex setting Figure 1 . Tissue engineering approaches were originally focused on regenerative medicine Langer and Vacanti, 1993 . In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 .", "In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 . The adaptability of these tissue-engineered models to multiple pathogens suggests a great potential for studies of infectious diseases. For instance, the lung tissue model relevant for pneumonia consists of lung fibroblasts embedded in a collagen matrix with a stratified epithelial layer on top . . The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 .", ". The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 . A recent publication demonstrated a two-hit-event of lung pathology in staphylococcal necrotizing pneumonia . . While the α-toxin had direct damaging effect on the lung epithelium, PVL induced lung pathology indirectly through the lysis of neutrophils . . All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 .", ". All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 . On these terms, the use of cultured ex vivo human organ biopsies, which are rare due to ethical considerations, is an additional option to study host-pathogen interactions. This ex vivo system may overcome even the limitations of the engineered tissue. In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae .", "In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae . , and IAV Nicholls et al., 2007; Chan et al., 2009 , were performed. In the human setting, most of the work focused on tropism, severity of infections, release of inflammatory mediators, and replication rates of the microorganisms. In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage .", "In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage . . However, this is just a beginning and more investigations are needed to unravel the complexity underlying these highly invasive infections. In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm.", "In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm. Despite many advances in recent years, more knowledge on mechanisms and immunology of disease progression is needed. The synergistic mechanisms between viruses and bacteria leading to enhanced morbidity and mortality are poorly understood. In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates.", "In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates. However, all have limitations. Here, we suggest using the porcine model, which provides obvious advantages in studies of human infectious diseases and should be considered much more frequent for future studies on severe infectious diseases, including pneumonia." ]

[ "Bacterial and viral co-infections of the respiratory tract are life-threatening and present a global burden to the global community. Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes are frequent colonizers of the upper respiratory tract. Imbalances through acquisition of seasonal viruses, e.g., Influenza A virus, can lead to bacterial dissemination to the lower respiratory tract, which in turn can result in severe pneumonia. In this review, we summarize the current knowledge about bacterial and viral co-infections of the respiratory tract and focus on potential experimental models suitable for mimicking this disease. Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available.", "Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available. The knowledge gained from these studies led to important discoveries and advances in understanding these infectious diseases. Nevertheless, mouse and other infection models have limitations, especially in translation of the discoveries to humans. Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 .", "Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 . The upper respiratory tract is a natural niche for potentially pathogenic bacteria embedded in commensal communities forming the nasopharyngeal microbiome. In particular, the microbial communities of the nasopharynx . are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . .", "are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . . The composition of the nasopharyngeal microbiome is highly dynamic Biesbroek et al., 2014a,b,c and many factors, including environmental and host factors, can affect microbial colonization . . Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b .", "Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b . The dynamic microbiome composition is guaranteed through the interplay between bacterial species, other microbes, and changing environmental conditions, as well as host-bacteria interactions Blaser and Falkow, 2009 . Most of the time, the microbiome and its interplay with the human host are believed to be beneficial for both Pettigrew et al., 2008; Murphy et al., 2009 . However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues .", "However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues . . While commensal respiratory microbiota facilitated immune-support against Influenza A virus infection IAV , oral treatment with antibiotics resulted not only in a shift of bacterial composition, but also in impaired CD4 T-, CD8 T-, and B-cell immunity following infection with IAV in mice . . Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls .", ". Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls . . However, another study published in the same year contradicted these results . . Chaban and colleagues analyzed microbiomes of 65 patients from H1N1 IAV outbreak in 2009. Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . .", "Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . . In this review we discuss secondary bacterial infections of the respiratory tract after primary infection by IAV with a focus on mechanisms by which these interactions are potentially mediated, and we will provide insight into the host contribution and immunological consequences. We further focus on potential animal models suitable for mimicking asymptomatic bacterial colonization and disease progression and thus, enabling to study adaptation strategies, viral-bacterial interactions, and immune responses in these highly lethal co-infections. Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . .", "Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . . The 8-segmented genomes of influenza A viruses are characterized by a significant plasticity. Due to point mutations and re-assortment events new variants or strains with epidemic or pandemic potential emerge Neumann et al., 2009 . In addition, influenza can be transmitted between animals, including swine, birds, horses, and humans, making it a zoonotic disease . . Seasonal influenza usually resolves without consequences in healthy individuals.", ". Seasonal influenza usually resolves without consequences in healthy individuals. However, it is estimated that seasonal influenza effects 5-10% of the world's population resulting in about 250,000 to 500,000 deaths annually Tjon-Kon-Fat et al., 2016 . At greater risk to develop secondary bacterial pneumonia are individuals with comorbidities, elderly people age > 65 , pregnant women, and children under the age of one . . For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 .", ". For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 . The pandemic killed around 50 million people worldwide and remains unique in its severity compared to other big outbreaks. However, many of the findings have been reinterpreted in recent years Brundage and Shanks, 2007; Chien et al., 2009 . It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . .", "It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . . Individual studies limited to certain regions identified also other pathogens commonly colonizing the respiratory tract, including Staphylococcus aureus, group A streptococcus GAS and Haemophilus influenzae Brundage and Shanks, 2008 . During the next two pandemics H2N2 Asian Flu 1957 and H3N2 Hong Kong Flu 1968 −1969 bacterial co-infections were less likely the cause of death compared to the Spanish Flu Giles and Shuttleworth, 1957; Trotter et al., 1959 . Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 .", "Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 . In 1957-1958, S. aureus was predominantly isolated from fatal pneumonia cases Hers et al., 1957 Hers et al., , 1958 Robertson et al., 1958; Martin et al., 1959 , whereas S. pneumoniae returned as predominant cause of severe pneumonia during the Hong Kong Flu Sharrar, 1969; Bisno et al., 1971; Burk et al., 1971; Schwarzmann et al., 1971 . Forty years later in 2009, a novel H1N1 virus of swine origin emerged and caused again a pandemic Dawood et al., 2009 Dawood et al., , 2012 . In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 .", "In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 . Although regional variations occurred, pneumococci and S. aureus were the most frequently isolated bacterial species Mauad et al., 2010; Shieh et al., 2010; Rice et al., 2012 . Group A streptococcus was absent in many local pneumonia outbreaks associated with viruses, but was predominant in others Brundage and Shanks, 2008; Ampofo et al., 2010 . When it does appear, it is typically third in incidence . .", "When it does appear, it is typically third in incidence . . Overall, data on pandemic outbreaks suggest that disease severity and mortality can be linked to secondary bacterial pathogens with variations depending on regions and state of immunity of the population Brundage and Shanks, 2008; Shanks et al., 2010 Shanks et al., , 2011 McCullers, 2013 . There is increasing evidence that the nasopharyngeal microbiota plays an important role in the pathogenesis of acute viral respiratory infections Teo et al., 2015; de Steenhuijsen Piters et al., 2016; Rosas-Salazar et al., 2016a,b . Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens.", "Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens. All of them are frequent colonizers of the human nasopharynx and they share many features including pathogenic mechanisms and clinical aspects Figure 1 . However, they also have unique properties. Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia.", "Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia. In the last decades, the pathogen became resistant to an increasing number of antibiotics and methicillin-resistant S. aureus MRSA is now a major cause of hospital acquired infections Hartman and Tomasz, 1984; Ubukata et al., 1989; Zetola et al., 2005 . Also the rise of community-acquired S. aureus strains is of special concern, because certain clones are associated with very severe infections . . Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 .", ". Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 . The pneumococcus is a typical colonizer of the human nasopharynx. About 20-50% of healthy children and 8-30% of healthy adults are asymptomatically colonized McCullers, 2006 . Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 .", "Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 . Group A streptococci colonize the mouth and upper respiratory tract in about 2-5% of world's population Okumura and Nizet, 2014 . The most common, non-invasive and mild infections caused by GAS are tonsillitis and pharyngitis with estimated 600 million cases per year . . Listed as number nine in the list of global killers with around 500,000 deaths annually .", ". Listed as number nine in the list of global killers with around 500,000 deaths annually . , it is obvious that this pathogen can cause severe invasive infections, including pneumonia, sepsis, streptococcal toxic shock syndrome, and necrotizing skin infections Cunningham, 2000; Carapetis et al., 2005 . Although all three pathogens are able to cause highly lethal diseases, the most fatal remains the pneumococcus, estimated to cause ca. 10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . .", "10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . . Influenza A virus binds via HA to either α2,3or α2,6-linked sialic acid at the surface of epithelial cells of the upper and lower respiratory tract . . Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways.", "Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways. Binding of viral RNA to retinoic acid inducible gene 1 induces the expression of type I and III interferons and activates transcription factor NF-κB, which in turn activates the release of pro-inflammatory cytokines Durbin et al., 2013; Iwasaki and Pillai, 2014 . In addition, inflammasome activation leads to the release of IL-1β and IL-18 Pothlichet et al., 2013; Iwasaki and Pillai, 2014 . All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 .", "All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 . Furthermore, aberrant coagulation induced by virus infection causes a hyper-inflammatory response Yang and Tang, 2016 . All these events contribute to lung tissue injury Imai et al., 2008; Davidson et al., 2014 . The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . .", "The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . . On the other hand, already colonized bacteria might enhance influenza virus virulence either by directly secreting proteases that cleave and activate HA Figure 2 Bottcher-Friebertshauser et al., 2013 or, indirectly, by activating host proteases such as plasminogen, which increases replication rates and infectivity of the virus Scheiblauer et al., 1992; Tse and Whittaker, 2015 . Potentially pathogenic bacteria, including the three species mentioned above, express an arsenal of virulence factors responsible for attachment to human host structures. Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract.", "Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract. Seasonal IAV infection can lead to an increased risk of secondary bacterial infections, i.e., pneumonia. Several experimental models can be used for studying these severe infections. Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases.", "Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases. In vivo bacterial and viral co-infections are mainly performed in mice, which does not necessarily resemble the human physiology and immune system. Thus, we suggest using the porcine model, which nearly resembles over 80% of the human immune system. MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 .", "MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 . Once the initial attachment occurs, bacterial cytotoxins including pneumolysin of pneumococci Garcia-Suarez Mdel et al., 2007; Zahlten et al., 2015 , α-hemolysin and leukocidins of S. aureus . , and Streptolysins S and O and Streptococcal pyrogenic exotoxin B of S. pyogenes Tsai et al., 1998; Gurel et al., 2013; Siemens et al., 2015 Siemens et al., , 2016 , can synergize with viral counterparts to further increase lung tissue pathology. Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 .", "Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 . As the patient begins to recover from viral infection, secondary bacterial infections might occur . due to the incomplete wound healing and exposure of host membrane components, including laminin, collagens type I and IV to classical bacterial MSCRAMMs Louria et al., 1959; Puchelle et al., 2006 . Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 .", "Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 . Respiratory viruses like IAV are able to induce suppression and killing of the resident alveolar macrophages Figure 2 . . These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections .", "These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections . . In addition, induction of interferons as a response to viral infection compromises the immune sensing of Gram-positive bacteria by neutrophils and macrophages, which would normally clear the bacteria from the lungs Figure 2 Sun and Metzger, 2008; Tian et al., 2012 . The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state .", "The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state . . The subsequent infection with Gram-positive bacteria, e.g., pneumococci, enhances the type I interferon expression, which in turn suppresses production of the CCL2 chemokine and recruitment of macrophages Nakamura Frontiers in Microbiology | FIGURE 2 | The interplay between IAV, bacteria, and the human host. The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment.", "The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment. IAV is able to induce suppression and killing of resident alveolar macrophages AM , which in turn delays viral clearance. The release of viral RNA activates different immune response pathways resulting in cytokine storm. Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages.", "Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages. After initial inflammation, the situation might worsen due to cellular infiltration of the lungs by neutrophils PMN , leading to an increased degranulation and tissue damage by effector molecules, including heparin-binding protein HBP . et al., 2011 . Another study by Shahangian et al. . revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion.", ". revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion. Other studies found that IAV exposed lungs had impaired natural killer NK cell responses in the airway to subsequent S. aureus infection . . Reduced TNFα production by NK cells was identified as a crucial upstream mechanism of depressed antimicrobial activities by alveolar macrophages Figure 2 . . It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . .", "It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . . In vitro studies on the interplay between IAV-pneumococci and human dendritic cells revealed TLR3 as a crucial sensor of viral and bacterial RNA leading to enhanced IL-12p70 production, which in turn might promote an anti-viral state by upregulation of interferons Yamamoto et al., 2004; Spelmink et al., 2016 . However, it should be noted that depending on the bacterial species the disease manifestation and underlying innate immune responses might vary Sharma-Chawla et al., 2016 . A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 .", "A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 . These processes are characterized by a general anti-inflammatory state and suppression of mechanisms involved in pathogen clearance due to increased interleukin-10 production van der Sluijs et al., 2004; Metzger and Sun, 2013 . The anti-inflammatory state suppresses the expression of pattern recognition receptors PRR on professional phagocytes leading to impaired phagocytosis and killing of microbes. These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis .", "These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis . . In co-infections, after initial inflammation in response to viral infection the situation might worsen due to bacterial invasion and enhanced cellular infiltration of the lungs by neutrophils, leading to an increased tissue damage and cytokine storm Figure 2 Conenello et al., 2007; McAuley et al., 2007 McAuley et al., , 2010 Porto and Stein, 2016 . Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 .", "Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 . Bacterial pathogens also express a variety of cytolytic toxins that can contribute to inflammation and tissue pathology. Pneumolysin, a pneumococcal pore-forming toxin with low affinity to lung epithelial cells, can damage neutrophils by utilizing P2X7 receptor . . Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs .", ". Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs . . GAS toxins, including SLO and SpeB, are capable of directly causing tissue damage and promoting pro-inflammatory states through neutrophil lysis Snall et al., 2016; Uhlmann et al., 2016 . The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 .", "The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 . Figure 2 summarizes the interplay between virus, bacteria, and host. Experimental animal models are a useful tool to study in vivo effects of different infectious agents and they represent approximately 3% of all pneumonia research published in peerreview journals . . However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 .", ". However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 . Hackam and colleagues identified 2,000 articles published between 1980 and 2006 in seven leading scientific journals that regularly publish animal studies Hackam and Redelmeier, 2006 . Seventy-six out of 2,000 were highly cited with a median citation count of 889. Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 .", "Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 . In pneumonia models, mammalians are mostly used because of their anatomical and physiological proximity to humans . . To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms.", "To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms. Rapid reproductive rate, small size, less complicated handling, the ability to reproduce and compare results with already published bacterial and viral mono-infections, detailed knowledge of genetics and immune responses, and a plethora of available reagents to study infections in mice are reasons for the use of these animals. To avoid variations in responses due to genetic diversity inbred mice strains are useful tools for studies aiming to elucidate molecular mechanisms of diseases. In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 .", "In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 . However, mouse models for bacterial and/or viral infections have several limitations. Most of the bacterial and viral species under study are human pathogens. In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 .", "In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 . IAV was shown to be essential for pneumococcal transmission from colonized mice to their naive littermates and the transmission occurred only when all mice were infected with IAV . . et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 .", ". et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 . The infection of ferrets with human seasonal IAV isolates results in an upper respiratory tract infection similar to human influenza infection Tripp and Tompkins, 2009 . In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism.", "In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism. A report by Sanford and Ramsay showed enhanced staphylococcal colonization of the upper respiratory tract in IAV infected animals as compared to non-infected, while no difference between both groups was observed in group B streptococcal infection Sanford and Ramsay, 1987 . In contrast, Smith and Mc Cullers reported lack of establishment of staphylococcal infection even when ferrets were pre-infected with IAV Smith and McCullers, 2014 . The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 .", "The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 . In recent years, the guinea pig Cavia porcellus was also used in pneumonia research. The physiology and anatomy of the guinea pig lung resembles to a certain extent the human lung and this model organism is often used in non-infectious lung diseases, including asthma and chronic obstructive pulmonary disease Canning and Chou, 2008 . In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses.", "In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses. Although viral replication can be readily detected upon intranasal inoculation in the upper respiratory tract and the lungs, guinea pigs exhibit only minor clinical symptoms Lowen et al., 2006; Gabbard et al., 2014 . However, the lung pathology of human IAV infected guinea pigs correlates with the clinical severity of human infection . . Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . .", ". Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . . Guinea pigs infected with pneumococci alone and cage-mated with non-treated contact animals transmitted the bacteria only in 7% of cases, while Sendai-virus infected, co-housed guinea pigs acquired pneumococcal infection in 83% of contacts . . Another study evaluated antibiotic efficacy in invasive pulmonary infection caused by penicillin resistant pneumococcus . . Intratracheal instillation of 3 × 10 9 CFU of S. pneumoniae induced a fatal pneumonia and bacteremia in 85% of untreated animals within 46 h . . As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents.", ". As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents. This is in parts due to the lack of species specific reagents, which is a disadvantage in using this model organism. Recently, the cotton rat Sigmodon hispidus was reported to be susceptible to IAV. Nasal and pulmonary infection in adult inbred cotton rats did not require viral adaptation . . The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days.", "The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days. Tissue pathology included damage of bronchiolar epithelium and the animals developed pneumonia which persisted for nearly 3 weeks . . In bacteriological studies rats are more frequently used. There are numerous rat models investigating the impact of diabetes . , metabolic syndromes . , cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors .", ", cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors . on development of pneumococcal, streptococcal, and staphylococcal pneumonia and lung pathology. Unfortunately, there are only few studies on bacterial and viral co-infections in rats. The first was performed by Harford et al., 1946 Harford et al., 1946 . The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . .", "The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . . Another study on human respiratory syncytial virus and S. pneumoniae revealed that rats were easily colonized with pneumococci, but viral replication after subsequent infection was strain dependent. In addition, neither pneumococci nor the virus spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates . Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents.", "Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents. Rabbits Oryctolagus cuniculus are well known for their use in studying cardiovascular diseases, antibody production, and eye research. Rabbits were also employed to study pneumonia, although only a few models are available. Typical read-out parameters include survival, leukocyte infiltration of the lungs, lung pathology, and assessment of drug concentration in serum. One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . .", "One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . . This study revealed that rabbits possess an active immunity if they have recovered from one attack of experimental pneumonia and they may subsequently resist repeated intra-tracheal dosages of pneumococci Kline and Winternitz, 1913 . In 1926 an infection by inhalation of Type I pneumococci was established in rabbits Stillman and Branch, 1926 . The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 .", "The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 . However, this infection route requires surgery and species-specific reagents are scarce. In IAV research rabbits are frequently used for antibody production and for studies on antibody kinetics following single or multiple IAV administrations Loza-Tulimowska et al., 1977 . Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp.", "Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp. was investigated revealing that nasally and orally inoculated cottontails shed relatively large quantities of viral RNA . . Notably, low viral titers were found to be sufficient to initiate viral replication in cottontails . . However, despite their susceptibility to IAV infection, rabbits are only rarely used as model for IAV pathogenesis since they offer no improvement over other established infection models. Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable.", "Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable. The species most commonly used are rhesus macaques Macaca mulatta and cynomolgus macaques Macaca fasciluraris . Although it was shown early that macaques were susceptible to IAV . , the animal models of choice remained ferrets and mice. Recently, macaques have been used to compare the pathogenesis of highly virulent 1918 pandemic IAV and the pathogenic bird flu strain H5N1 with a conventional H1N1 strain . . Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . .", ". Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . . The 1918 IAV strain induced dysregulation of the antiviral response leading to insufficient protection of the host, which in turn resulted in acute respiratory distress and a fatal outcome . . The 2009 pandemic H1N1 US isolate caused severe pathological lesions in the lungs of the macaques Itoh et al., 2009 . The three studies mentioned above used combined intratracheal delivery of high doses of virus. A recent study by Marriott et al. analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . .", "analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . . Virus replication was detected in all challenge groups, although the disease remained sub-clinical. In bacteriological studies non-human primates are rarely used. For group A streptococcal infection longitudinal transcriptome analyses were performed in experimental pharyngitis . and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a .", "and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a . Another study by Olsen and colleagues analyzed the contribution of PVL of a highly virulent USA300 S. aureus strain in respiratory infection Olsen et al., 2010b . Although the lower respiratory tract disease observed in monkey mimicked the clinical and pathological features of early mild to moderate pneumonia in humans, no involvement of PVL in lung pathology or immune cell influx of the lungs could be detected Olsen et al., 2010b . The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . .", "The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . . Pneumonia progression was monitored by clinical parameters assessment, blood chemistry, nasal swabs, and pathology of the lungs. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but did not predispose the animals to subsequent severe infection with the USA300 clone . . Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade.", "Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade. In 2013, Philipp and colleagues analyzed the carriage rate of pneumococcus in 158 colony animals. None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci.", "None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci. But, when infants were colonized with 19F strain via nasopharyngeal instillation, the colonization was induced in eight of eight infants, lasted for 2 weeks in all animals and for 7 weeks in more than 60% . . The same group tested detoxified pneumolysin dPly and pneumococcal histidine triad protein D PhtD as potential vaccine candidates to prevent pneumonia . . After immunization the rhesus macaques were challenged with a 19F pneumococcal strain.", ". After immunization the rhesus macaques were challenged with a 19F pneumococcal strain. AS02-adjuvanted PhtD-dPly vaccine protected the animals against S. pneumoniae-induced pneumonia, which was linked to the capacity i to greatly reduce bacterial load within the first week post-challenge and ii the levels of PhtD-and Ply-specific antibodies . . Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified.", "Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified. Small mammals including rodents are well known from a biological, genetic, and immunological point of view and are easy to maintain. The choice of these particular animals for infectious disease studies is often a result of a compromise between technical and financial options. However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans.", "However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans. Primates are usually legally reserved to specific topics. In this case, pigs could be an appropriate model system for studying infectious diseases including pneumonia Figure 1 . The composition and size of the porcine genome is comparable to that of humans . . In addition, human and porcine organs have many common features and functions . . The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar.", ". The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar. Furthermore, like humans, pigs possess tonsils, which are absent in mice . . A major advantage of studying infectious diseases by utilizing pigs as a host organism is that pigs have a full set of innate and adaptive immune effectors. According to whole genome sequencing results the porcine immune system resembles over 80% of the human immune system, whereas mice share less than 10% with humans . . Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 .", ". Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 . In contrast to mice and similar to humans, pigs have 50-70% of circulating polymorph nuclear cells . . In addition, all functional cytokines or orthologs involved in Th1, Th2, Th17, and Treg paradigm and corresponding immune cells have been described in pigs Murtaugh et al., 2009; Kaser et al., 2011; Kiros et al., 2011 . Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . .", "Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . . In contrast to human monocytes, which can be divided in three subclasses classical CD14 + CD16 − , nonclassical CD14 + CD16 + , and intermediate CD14 ++ CD16 + , porcine monocytes consist of four subclasses Chamorro et al., 2005; Fairbairn et al., 2013 . Like human monocytes they express adhesion molecules, such as VLA-4 and LFA-1 and costimulatory molecules, including CD80 and CD86 . . The pig has previously been used to mimic a number of human infectious diseases.", ". The pig has previously been used to mimic a number of human infectious diseases. Examples for S. aureus infections with this model organism are wound infections Svedman et al., 1989 , osteomyelitis . , and sepsis . . Intravenous inoculation of piglets with pneumococci led to bacteremia during a 5 days period and was associated with fever and septic arthritis. Intranasal inoculation of piglets led to colonization for at least six consecutive days without causing clinical signs . . In addition, research on respiratory infections of pigs by human pathogens including S. aureus . , Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years.", ", Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years. The fact that pigs and humans are infected with identical subtypes of IAV H1N1, H3N2 , and show similar clinical presentation and pathogenesis, makes pigs an ideal model organism for studies on respiratory co-infections . . Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis.", "Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis. S. suis usually inhabits mucosal surfaces of tonsils, nares, genital and alimentary tract of piglets. Once the microbial balance is disturbed, the bacteria can cause meningitis, septicemia, arthritis, and pneumonia in pigs . . Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent.", ". Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent. In general, serotype 2 is most frequently isolated from diseased pigs . . S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 .", "S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 . A recent publication by Lin and colleagues on H1N1 and S. suis co-infected piglets demonstrated the synergistic effects of both pathogens . . Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . .", ". Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . . In addition, genes associated with immune responses, inflammatory cytokine production, and apoptotic pathways were highly overexpressed in the coinfected group . . Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals.", ". Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals. Although the use of animals contributes greatly to our understanding of infectious diseases, human 3D-organotypic tissue models and ex vivo organ tissues should be considered, as they are most valuable tools to study host-pathogen interactions in a more complex setting Figure 1 . Tissue engineering approaches were originally focused on regenerative medicine Langer and Vacanti, 1993 . In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 .", "In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 . The adaptability of these tissue-engineered models to multiple pathogens suggests a great potential for studies of infectious diseases. For instance, the lung tissue model relevant for pneumonia consists of lung fibroblasts embedded in a collagen matrix with a stratified epithelial layer on top . . The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 .", ". The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 . A recent publication demonstrated a two-hit-event of lung pathology in staphylococcal necrotizing pneumonia . . While the α-toxin had direct damaging effect on the lung epithelium, PVL induced lung pathology indirectly through the lysis of neutrophils . . All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 .", ". All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 . On these terms, the use of cultured ex vivo human organ biopsies, which are rare due to ethical considerations, is an additional option to study host-pathogen interactions. This ex vivo system may overcome even the limitations of the engineered tissue. In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae .", "In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae . , and IAV Nicholls et al., 2007; Chan et al., 2009 , were performed. In the human setting, most of the work focused on tropism, severity of infections, release of inflammatory mediators, and replication rates of the microorganisms. In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage .", "In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage . . However, this is just a beginning and more investigations are needed to unravel the complexity underlying these highly invasive infections. In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm.", "In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm. Despite many advances in recent years, more knowledge on mechanisms and immunology of disease progression is needed. The synergistic mechanisms between viruses and bacteria leading to enhanced morbidity and mortality are poorly understood. In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates.", "In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates. However, all have limitations. Here, we suggest using the porcine model, which provides obvious advantages in studies of human infectious diseases and should be considered much more frequent for future studies on severe infectious diseases, including pneumonia." ]

[ "Bacterial and viral co-infections of the respiratory tract are life-threatening and present a global burden to the global community. Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes are frequent colonizers of the upper respiratory tract. Imbalances through acquisition of seasonal viruses, e.g., Influenza A virus, can lead to bacterial dissemination to the lower respiratory tract, which in turn can result in severe pneumonia. In this review, we summarize the current knowledge about bacterial and viral co-infections of the respiratory tract and focus on potential experimental models suitable for mimicking this disease. Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available.", "Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available. The knowledge gained from these studies led to important discoveries and advances in understanding these infectious diseases. Nevertheless, mouse and other infection models have limitations, especially in translation of the discoveries to humans. Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 .", "Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 . The upper respiratory tract is a natural niche for potentially pathogenic bacteria embedded in commensal communities forming the nasopharyngeal microbiome. In particular, the microbial communities of the nasopharynx . are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . .", "are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . . The composition of the nasopharyngeal microbiome is highly dynamic Biesbroek et al., 2014a,b,c and many factors, including environmental and host factors, can affect microbial colonization . . Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b .", "Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b . The dynamic microbiome composition is guaranteed through the interplay between bacterial species, other microbes, and changing environmental conditions, as well as host-bacteria interactions Blaser and Falkow, 2009 . Most of the time, the microbiome and its interplay with the human host are believed to be beneficial for both Pettigrew et al., 2008; Murphy et al., 2009 . However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues .", "However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues . . While commensal respiratory microbiota facilitated immune-support against Influenza A virus infection IAV , oral treatment with antibiotics resulted not only in a shift of bacterial composition, but also in impaired CD4 T-, CD8 T-, and B-cell immunity following infection with IAV in mice . . Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls .", ". Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls . . However, another study published in the same year contradicted these results . . Chaban and colleagues analyzed microbiomes of 65 patients from H1N1 IAV outbreak in 2009. Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . .", "Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . . In this review we discuss secondary bacterial infections of the respiratory tract after primary infection by IAV with a focus on mechanisms by which these interactions are potentially mediated, and we will provide insight into the host contribution and immunological consequences. We further focus on potential animal models suitable for mimicking asymptomatic bacterial colonization and disease progression and thus, enabling to study adaptation strategies, viral-bacterial interactions, and immune responses in these highly lethal co-infections. Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . .", "Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . . The 8-segmented genomes of influenza A viruses are characterized by a significant plasticity. Due to point mutations and re-assortment events new variants or strains with epidemic or pandemic potential emerge Neumann et al., 2009 . In addition, influenza can be transmitted between animals, including swine, birds, horses, and humans, making it a zoonotic disease . . Seasonal influenza usually resolves without consequences in healthy individuals.", ". Seasonal influenza usually resolves without consequences in healthy individuals. However, it is estimated that seasonal influenza effects 5-10% of the world's population resulting in about 250,000 to 500,000 deaths annually Tjon-Kon-Fat et al., 2016 . At greater risk to develop secondary bacterial pneumonia are individuals with comorbidities, elderly people age > 65 , pregnant women, and children under the age of one . . For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 .", ". For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 . The pandemic killed around 50 million people worldwide and remains unique in its severity compared to other big outbreaks. However, many of the findings have been reinterpreted in recent years Brundage and Shanks, 2007; Chien et al., 2009 . It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . .", "It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . . Individual studies limited to certain regions identified also other pathogens commonly colonizing the respiratory tract, including Staphylococcus aureus, group A streptococcus GAS and Haemophilus influenzae Brundage and Shanks, 2008 . During the next two pandemics H2N2 Asian Flu 1957 and H3N2 Hong Kong Flu 1968 −1969 bacterial co-infections were less likely the cause of death compared to the Spanish Flu Giles and Shuttleworth, 1957; Trotter et al., 1959 . Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 .", "Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 . In 1957-1958, S. aureus was predominantly isolated from fatal pneumonia cases Hers et al., 1957 Hers et al., , 1958 Robertson et al., 1958; Martin et al., 1959 , whereas S. pneumoniae returned as predominant cause of severe pneumonia during the Hong Kong Flu Sharrar, 1969; Bisno et al., 1971; Burk et al., 1971; Schwarzmann et al., 1971 . Forty years later in 2009, a novel H1N1 virus of swine origin emerged and caused again a pandemic Dawood et al., 2009 Dawood et al., , 2012 . In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 .", "In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 . Although regional variations occurred, pneumococci and S. aureus were the most frequently isolated bacterial species Mauad et al., 2010; Shieh et al., 2010; Rice et al., 2012 . Group A streptococcus was absent in many local pneumonia outbreaks associated with viruses, but was predominant in others Brundage and Shanks, 2008; Ampofo et al., 2010 . When it does appear, it is typically third in incidence . .", "When it does appear, it is typically third in incidence . . Overall, data on pandemic outbreaks suggest that disease severity and mortality can be linked to secondary bacterial pathogens with variations depending on regions and state of immunity of the population Brundage and Shanks, 2008; Shanks et al., 2010 Shanks et al., , 2011 McCullers, 2013 . There is increasing evidence that the nasopharyngeal microbiota plays an important role in the pathogenesis of acute viral respiratory infections Teo et al., 2015; de Steenhuijsen Piters et al., 2016; Rosas-Salazar et al., 2016a,b . Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens.", "Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens. All of them are frequent colonizers of the human nasopharynx and they share many features including pathogenic mechanisms and clinical aspects Figure 1 . However, they also have unique properties. Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia.", "Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia. In the last decades, the pathogen became resistant to an increasing number of antibiotics and methicillin-resistant S. aureus MRSA is now a major cause of hospital acquired infections Hartman and Tomasz, 1984; Ubukata et al., 1989; Zetola et al., 2005 . Also the rise of community-acquired S. aureus strains is of special concern, because certain clones are associated with very severe infections . . Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 .", ". Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 . The pneumococcus is a typical colonizer of the human nasopharynx. About 20-50% of healthy children and 8-30% of healthy adults are asymptomatically colonized McCullers, 2006 . Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 .", "Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 . Group A streptococci colonize the mouth and upper respiratory tract in about 2-5% of world's population Okumura and Nizet, 2014 . The most common, non-invasive and mild infections caused by GAS are tonsillitis and pharyngitis with estimated 600 million cases per year . . Listed as number nine in the list of global killers with around 500,000 deaths annually .", ". Listed as number nine in the list of global killers with around 500,000 deaths annually . , it is obvious that this pathogen can cause severe invasive infections, including pneumonia, sepsis, streptococcal toxic shock syndrome, and necrotizing skin infections Cunningham, 2000; Carapetis et al., 2005 . Although all three pathogens are able to cause highly lethal diseases, the most fatal remains the pneumococcus, estimated to cause ca. 10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . .", "10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . . Influenza A virus binds via HA to either α2,3or α2,6-linked sialic acid at the surface of epithelial cells of the upper and lower respiratory tract . . Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways.", "Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways. Binding of viral RNA to retinoic acid inducible gene 1 induces the expression of type I and III interferons and activates transcription factor NF-κB, which in turn activates the release of pro-inflammatory cytokines Durbin et al., 2013; Iwasaki and Pillai, 2014 . In addition, inflammasome activation leads to the release of IL-1β and IL-18 Pothlichet et al., 2013; Iwasaki and Pillai, 2014 . All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 .", "All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 . Furthermore, aberrant coagulation induced by virus infection causes a hyper-inflammatory response Yang and Tang, 2016 . All these events contribute to lung tissue injury Imai et al., 2008; Davidson et al., 2014 . The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . .", "The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . . On the other hand, already colonized bacteria might enhance influenza virus virulence either by directly secreting proteases that cleave and activate HA Figure 2 Bottcher-Friebertshauser et al., 2013 or, indirectly, by activating host proteases such as plasminogen, which increases replication rates and infectivity of the virus Scheiblauer et al., 1992; Tse and Whittaker, 2015 . Potentially pathogenic bacteria, including the three species mentioned above, express an arsenal of virulence factors responsible for attachment to human host structures. Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract.", "Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract. Seasonal IAV infection can lead to an increased risk of secondary bacterial infections, i.e., pneumonia. Several experimental models can be used for studying these severe infections. Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases.", "Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases. In vivo bacterial and viral co-infections are mainly performed in mice, which does not necessarily resemble the human physiology and immune system. Thus, we suggest using the porcine model, which nearly resembles over 80% of the human immune system. MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 .", "MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 . Once the initial attachment occurs, bacterial cytotoxins including pneumolysin of pneumococci Garcia-Suarez Mdel et al., 2007; Zahlten et al., 2015 , α-hemolysin and leukocidins of S. aureus . , and Streptolysins S and O and Streptococcal pyrogenic exotoxin B of S. pyogenes Tsai et al., 1998; Gurel et al., 2013; Siemens et al., 2015 Siemens et al., , 2016 , can synergize with viral counterparts to further increase lung tissue pathology. Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 .", "Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 . As the patient begins to recover from viral infection, secondary bacterial infections might occur . due to the incomplete wound healing and exposure of host membrane components, including laminin, collagens type I and IV to classical bacterial MSCRAMMs Louria et al., 1959; Puchelle et al., 2006 . Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 .", "Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 . Respiratory viruses like IAV are able to induce suppression and killing of the resident alveolar macrophages Figure 2 . . These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections .", "These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections . . In addition, induction of interferons as a response to viral infection compromises the immune sensing of Gram-positive bacteria by neutrophils and macrophages, which would normally clear the bacteria from the lungs Figure 2 Sun and Metzger, 2008; Tian et al., 2012 . The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state .", "The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state . . The subsequent infection with Gram-positive bacteria, e.g., pneumococci, enhances the type I interferon expression, which in turn suppresses production of the CCL2 chemokine and recruitment of macrophages Nakamura Frontiers in Microbiology | FIGURE 2 | The interplay between IAV, bacteria, and the human host. The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment.", "The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment. IAV is able to induce suppression and killing of resident alveolar macrophages AM , which in turn delays viral clearance. The release of viral RNA activates different immune response pathways resulting in cytokine storm. Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages.", "Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages. After initial inflammation, the situation might worsen due to cellular infiltration of the lungs by neutrophils PMN , leading to an increased degranulation and tissue damage by effector molecules, including heparin-binding protein HBP . et al., 2011 . Another study by Shahangian et al. . revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion.", ". revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion. Other studies found that IAV exposed lungs had impaired natural killer NK cell responses in the airway to subsequent S. aureus infection . . Reduced TNFα production by NK cells was identified as a crucial upstream mechanism of depressed antimicrobial activities by alveolar macrophages Figure 2 . . It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . .", "It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . . In vitro studies on the interplay between IAV-pneumococci and human dendritic cells revealed TLR3 as a crucial sensor of viral and bacterial RNA leading to enhanced IL-12p70 production, which in turn might promote an anti-viral state by upregulation of interferons Yamamoto et al., 2004; Spelmink et al., 2016 . However, it should be noted that depending on the bacterial species the disease manifestation and underlying innate immune responses might vary Sharma-Chawla et al., 2016 . A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 .", "A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 . These processes are characterized by a general anti-inflammatory state and suppression of mechanisms involved in pathogen clearance due to increased interleukin-10 production van der Sluijs et al., 2004; Metzger and Sun, 2013 . The anti-inflammatory state suppresses the expression of pattern recognition receptors PRR on professional phagocytes leading to impaired phagocytosis and killing of microbes. These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis .", "These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis . . In co-infections, after initial inflammation in response to viral infection the situation might worsen due to bacterial invasion and enhanced cellular infiltration of the lungs by neutrophils, leading to an increased tissue damage and cytokine storm Figure 2 Conenello et al., 2007; McAuley et al., 2007 McAuley et al., , 2010 Porto and Stein, 2016 . Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 .", "Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 . Bacterial pathogens also express a variety of cytolytic toxins that can contribute to inflammation and tissue pathology. Pneumolysin, a pneumococcal pore-forming toxin with low affinity to lung epithelial cells, can damage neutrophils by utilizing P2X7 receptor . . Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs .", ". Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs . . GAS toxins, including SLO and SpeB, are capable of directly causing tissue damage and promoting pro-inflammatory states through neutrophil lysis Snall et al., 2016; Uhlmann et al., 2016 . The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 .", "The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 . Figure 2 summarizes the interplay between virus, bacteria, and host. Experimental animal models are a useful tool to study in vivo effects of different infectious agents and they represent approximately 3% of all pneumonia research published in peerreview journals . . However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 .", ". However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 . Hackam and colleagues identified 2,000 articles published between 1980 and 2006 in seven leading scientific journals that regularly publish animal studies Hackam and Redelmeier, 2006 . Seventy-six out of 2,000 were highly cited with a median citation count of 889. Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 .", "Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 . In pneumonia models, mammalians are mostly used because of their anatomical and physiological proximity to humans . . To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms.", "To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms. Rapid reproductive rate, small size, less complicated handling, the ability to reproduce and compare results with already published bacterial and viral mono-infections, detailed knowledge of genetics and immune responses, and a plethora of available reagents to study infections in mice are reasons for the use of these animals. To avoid variations in responses due to genetic diversity inbred mice strains are useful tools for studies aiming to elucidate molecular mechanisms of diseases. In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 .", "In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 . However, mouse models for bacterial and/or viral infections have several limitations. Most of the bacterial and viral species under study are human pathogens. In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 .", "In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 . IAV was shown to be essential for pneumococcal transmission from colonized mice to their naive littermates and the transmission occurred only when all mice were infected with IAV . . et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 .", ". et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 . The infection of ferrets with human seasonal IAV isolates results in an upper respiratory tract infection similar to human influenza infection Tripp and Tompkins, 2009 . In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism.", "In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism. A report by Sanford and Ramsay showed enhanced staphylococcal colonization of the upper respiratory tract in IAV infected animals as compared to non-infected, while no difference between both groups was observed in group B streptococcal infection Sanford and Ramsay, 1987 . In contrast, Smith and Mc Cullers reported lack of establishment of staphylococcal infection even when ferrets were pre-infected with IAV Smith and McCullers, 2014 . The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 .", "The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 . In recent years, the guinea pig Cavia porcellus was also used in pneumonia research. The physiology and anatomy of the guinea pig lung resembles to a certain extent the human lung and this model organism is often used in non-infectious lung diseases, including asthma and chronic obstructive pulmonary disease Canning and Chou, 2008 . In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses.", "In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses. Although viral replication can be readily detected upon intranasal inoculation in the upper respiratory tract and the lungs, guinea pigs exhibit only minor clinical symptoms Lowen et al., 2006; Gabbard et al., 2014 . However, the lung pathology of human IAV infected guinea pigs correlates with the clinical severity of human infection . . Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . .", ". Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . . Guinea pigs infected with pneumococci alone and cage-mated with non-treated contact animals transmitted the bacteria only in 7% of cases, while Sendai-virus infected, co-housed guinea pigs acquired pneumococcal infection in 83% of contacts . . Another study evaluated antibiotic efficacy in invasive pulmonary infection caused by penicillin resistant pneumococcus . . Intratracheal instillation of 3 × 10 9 CFU of S. pneumoniae induced a fatal pneumonia and bacteremia in 85% of untreated animals within 46 h . . As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents.", ". As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents. This is in parts due to the lack of species specific reagents, which is a disadvantage in using this model organism. Recently, the cotton rat Sigmodon hispidus was reported to be susceptible to IAV. Nasal and pulmonary infection in adult inbred cotton rats did not require viral adaptation . . The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days.", "The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days. Tissue pathology included damage of bronchiolar epithelium and the animals developed pneumonia which persisted for nearly 3 weeks . . In bacteriological studies rats are more frequently used. There are numerous rat models investigating the impact of diabetes . , metabolic syndromes . , cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors .", ", cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors . on development of pneumococcal, streptococcal, and staphylococcal pneumonia and lung pathology. Unfortunately, there are only few studies on bacterial and viral co-infections in rats. The first was performed by Harford et al., 1946 Harford et al., 1946 . The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . .", "The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . . Another study on human respiratory syncytial virus and S. pneumoniae revealed that rats were easily colonized with pneumococci, but viral replication after subsequent infection was strain dependent. In addition, neither pneumococci nor the virus spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates . Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents.", "Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents. Rabbits Oryctolagus cuniculus are well known for their use in studying cardiovascular diseases, antibody production, and eye research. Rabbits were also employed to study pneumonia, although only a few models are available. Typical read-out parameters include survival, leukocyte infiltration of the lungs, lung pathology, and assessment of drug concentration in serum. One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . .", "One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . . This study revealed that rabbits possess an active immunity if they have recovered from one attack of experimental pneumonia and they may subsequently resist repeated intra-tracheal dosages of pneumococci Kline and Winternitz, 1913 . In 1926 an infection by inhalation of Type I pneumococci was established in rabbits Stillman and Branch, 1926 . The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 .", "The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 . However, this infection route requires surgery and species-specific reagents are scarce. In IAV research rabbits are frequently used for antibody production and for studies on antibody kinetics following single or multiple IAV administrations Loza-Tulimowska et al., 1977 . Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp.", "Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp. was investigated revealing that nasally and orally inoculated cottontails shed relatively large quantities of viral RNA . . Notably, low viral titers were found to be sufficient to initiate viral replication in cottontails . . However, despite their susceptibility to IAV infection, rabbits are only rarely used as model for IAV pathogenesis since they offer no improvement over other established infection models. Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable.", "Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable. The species most commonly used are rhesus macaques Macaca mulatta and cynomolgus macaques Macaca fasciluraris . Although it was shown early that macaques were susceptible to IAV . , the animal models of choice remained ferrets and mice. Recently, macaques have been used to compare the pathogenesis of highly virulent 1918 pandemic IAV and the pathogenic bird flu strain H5N1 with a conventional H1N1 strain . . Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . .", ". Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . . The 1918 IAV strain induced dysregulation of the antiviral response leading to insufficient protection of the host, which in turn resulted in acute respiratory distress and a fatal outcome . . The 2009 pandemic H1N1 US isolate caused severe pathological lesions in the lungs of the macaques Itoh et al., 2009 . The three studies mentioned above used combined intratracheal delivery of high doses of virus. A recent study by Marriott et al. analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . .", "analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . . Virus replication was detected in all challenge groups, although the disease remained sub-clinical. In bacteriological studies non-human primates are rarely used. For group A streptococcal infection longitudinal transcriptome analyses were performed in experimental pharyngitis . and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a .", "and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a . Another study by Olsen and colleagues analyzed the contribution of PVL of a highly virulent USA300 S. aureus strain in respiratory infection Olsen et al., 2010b . Although the lower respiratory tract disease observed in monkey mimicked the clinical and pathological features of early mild to moderate pneumonia in humans, no involvement of PVL in lung pathology or immune cell influx of the lungs could be detected Olsen et al., 2010b . The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . .", "The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . . Pneumonia progression was monitored by clinical parameters assessment, blood chemistry, nasal swabs, and pathology of the lungs. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but did not predispose the animals to subsequent severe infection with the USA300 clone . . Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade.", "Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade. In 2013, Philipp and colleagues analyzed the carriage rate of pneumococcus in 158 colony animals. None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci.", "None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci. But, when infants were colonized with 19F strain via nasopharyngeal instillation, the colonization was induced in eight of eight infants, lasted for 2 weeks in all animals and for 7 weeks in more than 60% . . The same group tested detoxified pneumolysin dPly and pneumococcal histidine triad protein D PhtD as potential vaccine candidates to prevent pneumonia . . After immunization the rhesus macaques were challenged with a 19F pneumococcal strain.", ". After immunization the rhesus macaques were challenged with a 19F pneumococcal strain. AS02-adjuvanted PhtD-dPly vaccine protected the animals against S. pneumoniae-induced pneumonia, which was linked to the capacity i to greatly reduce bacterial load within the first week post-challenge and ii the levels of PhtD-and Ply-specific antibodies . . Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified.", "Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified. Small mammals including rodents are well known from a biological, genetic, and immunological point of view and are easy to maintain. The choice of these particular animals for infectious disease studies is often a result of a compromise between technical and financial options. However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans.", "However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans. Primates are usually legally reserved to specific topics. In this case, pigs could be an appropriate model system for studying infectious diseases including pneumonia Figure 1 . The composition and size of the porcine genome is comparable to that of humans . . In addition, human and porcine organs have many common features and functions . . The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar.", ". The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar. Furthermore, like humans, pigs possess tonsils, which are absent in mice . . A major advantage of studying infectious diseases by utilizing pigs as a host organism is that pigs have a full set of innate and adaptive immune effectors. According to whole genome sequencing results the porcine immune system resembles over 80% of the human immune system, whereas mice share less than 10% with humans . . Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 .", ". Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 . In contrast to mice and similar to humans, pigs have 50-70% of circulating polymorph nuclear cells . . In addition, all functional cytokines or orthologs involved in Th1, Th2, Th17, and Treg paradigm and corresponding immune cells have been described in pigs Murtaugh et al., 2009; Kaser et al., 2011; Kiros et al., 2011 . Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . .", "Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . . In contrast to human monocytes, which can be divided in three subclasses classical CD14 + CD16 − , nonclassical CD14 + CD16 + , and intermediate CD14 ++ CD16 + , porcine monocytes consist of four subclasses Chamorro et al., 2005; Fairbairn et al., 2013 . Like human monocytes they express adhesion molecules, such as VLA-4 and LFA-1 and costimulatory molecules, including CD80 and CD86 . . The pig has previously been used to mimic a number of human infectious diseases.", ". The pig has previously been used to mimic a number of human infectious diseases. Examples for S. aureus infections with this model organism are wound infections Svedman et al., 1989 , osteomyelitis . , and sepsis . . Intravenous inoculation of piglets with pneumococci led to bacteremia during a 5 days period and was associated with fever and septic arthritis. Intranasal inoculation of piglets led to colonization for at least six consecutive days without causing clinical signs . . In addition, research on respiratory infections of pigs by human pathogens including S. aureus . , Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years.", ", Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years. The fact that pigs and humans are infected with identical subtypes of IAV H1N1, H3N2 , and show similar clinical presentation and pathogenesis, makes pigs an ideal model organism for studies on respiratory co-infections . . Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis.", "Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis. S. suis usually inhabits mucosal surfaces of tonsils, nares, genital and alimentary tract of piglets. Once the microbial balance is disturbed, the bacteria can cause meningitis, septicemia, arthritis, and pneumonia in pigs . . Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent.", ". Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent. In general, serotype 2 is most frequently isolated from diseased pigs . . S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 .", "S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 . A recent publication by Lin and colleagues on H1N1 and S. suis co-infected piglets demonstrated the synergistic effects of both pathogens . . Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . .", ". Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . . In addition, genes associated with immune responses, inflammatory cytokine production, and apoptotic pathways were highly overexpressed in the coinfected group . . Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals.", ". Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals. Although the use of animals contributes greatly to our understanding of infectious diseases, human 3D-organotypic tissue models and ex vivo organ tissues should be considered, as they are most valuable tools to study host-pathogen interactions in a more complex setting Figure 1 . Tissue engineering approaches were originally focused on regenerative medicine Langer and Vacanti, 1993 . In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 .", "In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 . The adaptability of these tissue-engineered models to multiple pathogens suggests a great potential for studies of infectious diseases. For instance, the lung tissue model relevant for pneumonia consists of lung fibroblasts embedded in a collagen matrix with a stratified epithelial layer on top . . The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 .", ". The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 . A recent publication demonstrated a two-hit-event of lung pathology in staphylococcal necrotizing pneumonia . . While the α-toxin had direct damaging effect on the lung epithelium, PVL induced lung pathology indirectly through the lysis of neutrophils . . All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 .", ". All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 . On these terms, the use of cultured ex vivo human organ biopsies, which are rare due to ethical considerations, is an additional option to study host-pathogen interactions. This ex vivo system may overcome even the limitations of the engineered tissue. In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae .", "In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae . , and IAV Nicholls et al., 2007; Chan et al., 2009 , were performed. In the human setting, most of the work focused on tropism, severity of infections, release of inflammatory mediators, and replication rates of the microorganisms. In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage .", "In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage . . However, this is just a beginning and more investigations are needed to unravel the complexity underlying these highly invasive infections. In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm.", "In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm. Despite many advances in recent years, more knowledge on mechanisms and immunology of disease progression is needed. The synergistic mechanisms between viruses and bacteria leading to enhanced morbidity and mortality are poorly understood. In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates.", "In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates. However, all have limitations. Here, we suggest using the porcine model, which provides obvious advantages in studies of human infectious diseases and should be considered much more frequent for future studies on severe infectious diseases, including pneumonia." ]

[ "Bacterial and viral co-infections of the respiratory tract are life-threatening and present a global burden to the global community. Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes are frequent colonizers of the upper respiratory tract. Imbalances through acquisition of seasonal viruses, e.g., Influenza A virus, can lead to bacterial dissemination to the lower respiratory tract, which in turn can result in severe pneumonia. In this review, we summarize the current knowledge about bacterial and viral co-infections of the respiratory tract and focus on potential experimental models suitable for mimicking this disease. Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available.", "Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available. The knowledge gained from these studies led to important discoveries and advances in understanding these infectious diseases. Nevertheless, mouse and other infection models have limitations, especially in translation of the discoveries to humans. Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 .", "Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 . The upper respiratory tract is a natural niche for potentially pathogenic bacteria embedded in commensal communities forming the nasopharyngeal microbiome. In particular, the microbial communities of the nasopharynx . are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . .", "are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . . The composition of the nasopharyngeal microbiome is highly dynamic Biesbroek et al., 2014a,b,c and many factors, including environmental and host factors, can affect microbial colonization . . Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b .", "Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b . The dynamic microbiome composition is guaranteed through the interplay between bacterial species, other microbes, and changing environmental conditions, as well as host-bacteria interactions Blaser and Falkow, 2009 . Most of the time, the microbiome and its interplay with the human host are believed to be beneficial for both Pettigrew et al., 2008; Murphy et al., 2009 . However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues .", "However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues . . While commensal respiratory microbiota facilitated immune-support against Influenza A virus infection IAV , oral treatment with antibiotics resulted not only in a shift of bacterial composition, but also in impaired CD4 T-, CD8 T-, and B-cell immunity following infection with IAV in mice . . Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls .", ". Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls . . However, another study published in the same year contradicted these results . . Chaban and colleagues analyzed microbiomes of 65 patients from H1N1 IAV outbreak in 2009. Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . .", "Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . . In this review we discuss secondary bacterial infections of the respiratory tract after primary infection by IAV with a focus on mechanisms by which these interactions are potentially mediated, and we will provide insight into the host contribution and immunological consequences. We further focus on potential animal models suitable for mimicking asymptomatic bacterial colonization and disease progression and thus, enabling to study adaptation strategies, viral-bacterial interactions, and immune responses in these highly lethal co-infections. Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . .", "Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . . The 8-segmented genomes of influenza A viruses are characterized by a significant plasticity. Due to point mutations and re-assortment events new variants or strains with epidemic or pandemic potential emerge Neumann et al., 2009 . In addition, influenza can be transmitted between animals, including swine, birds, horses, and humans, making it a zoonotic disease . . Seasonal influenza usually resolves without consequences in healthy individuals.", ". Seasonal influenza usually resolves without consequences in healthy individuals. However, it is estimated that seasonal influenza effects 5-10% of the world's population resulting in about 250,000 to 500,000 deaths annually Tjon-Kon-Fat et al., 2016 . At greater risk to develop secondary bacterial pneumonia are individuals with comorbidities, elderly people age > 65 , pregnant women, and children under the age of one . . For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 .", ". For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 . The pandemic killed around 50 million people worldwide and remains unique in its severity compared to other big outbreaks. However, many of the findings have been reinterpreted in recent years Brundage and Shanks, 2007; Chien et al., 2009 . It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . .", "It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . . Individual studies limited to certain regions identified also other pathogens commonly colonizing the respiratory tract, including Staphylococcus aureus, group A streptococcus GAS and Haemophilus influenzae Brundage and Shanks, 2008 . During the next two pandemics H2N2 Asian Flu 1957 and H3N2 Hong Kong Flu 1968 −1969 bacterial co-infections were less likely the cause of death compared to the Spanish Flu Giles and Shuttleworth, 1957; Trotter et al., 1959 . Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 .", "Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 . In 1957-1958, S. aureus was predominantly isolated from fatal pneumonia cases Hers et al., 1957 Hers et al., , 1958 Robertson et al., 1958; Martin et al., 1959 , whereas S. pneumoniae returned as predominant cause of severe pneumonia during the Hong Kong Flu Sharrar, 1969; Bisno et al., 1971; Burk et al., 1971; Schwarzmann et al., 1971 . Forty years later in 2009, a novel H1N1 virus of swine origin emerged and caused again a pandemic Dawood et al., 2009 Dawood et al., , 2012 . In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 .", "In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 . Although regional variations occurred, pneumococci and S. aureus were the most frequently isolated bacterial species Mauad et al., 2010; Shieh et al., 2010; Rice et al., 2012 . Group A streptococcus was absent in many local pneumonia outbreaks associated with viruses, but was predominant in others Brundage and Shanks, 2008; Ampofo et al., 2010 . When it does appear, it is typically third in incidence . .", "When it does appear, it is typically third in incidence . . Overall, data on pandemic outbreaks suggest that disease severity and mortality can be linked to secondary bacterial pathogens with variations depending on regions and state of immunity of the population Brundage and Shanks, 2008; Shanks et al., 2010 Shanks et al., , 2011 McCullers, 2013 . There is increasing evidence that the nasopharyngeal microbiota plays an important role in the pathogenesis of acute viral respiratory infections Teo et al., 2015; de Steenhuijsen Piters et al., 2016; Rosas-Salazar et al., 2016a,b . Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens.", "Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens. All of them are frequent colonizers of the human nasopharynx and they share many features including pathogenic mechanisms and clinical aspects Figure 1 . However, they also have unique properties. Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia.", "Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia. In the last decades, the pathogen became resistant to an increasing number of antibiotics and methicillin-resistant S. aureus MRSA is now a major cause of hospital acquired infections Hartman and Tomasz, 1984; Ubukata et al., 1989; Zetola et al., 2005 . Also the rise of community-acquired S. aureus strains is of special concern, because certain clones are associated with very severe infections . . Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 .", ". Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 . The pneumococcus is a typical colonizer of the human nasopharynx. About 20-50% of healthy children and 8-30% of healthy adults are asymptomatically colonized McCullers, 2006 . Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 .", "Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 . Group A streptococci colonize the mouth and upper respiratory tract in about 2-5% of world's population Okumura and Nizet, 2014 . The most common, non-invasive and mild infections caused by GAS are tonsillitis and pharyngitis with estimated 600 million cases per year . . Listed as number nine in the list of global killers with around 500,000 deaths annually .", ". Listed as number nine in the list of global killers with around 500,000 deaths annually . , it is obvious that this pathogen can cause severe invasive infections, including pneumonia, sepsis, streptococcal toxic shock syndrome, and necrotizing skin infections Cunningham, 2000; Carapetis et al., 2005 . Although all three pathogens are able to cause highly lethal diseases, the most fatal remains the pneumococcus, estimated to cause ca. 10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . .", "10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . . Influenza A virus binds via HA to either α2,3or α2,6-linked sialic acid at the surface of epithelial cells of the upper and lower respiratory tract . . Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways.", "Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways. Binding of viral RNA to retinoic acid inducible gene 1 induces the expression of type I and III interferons and activates transcription factor NF-κB, which in turn activates the release of pro-inflammatory cytokines Durbin et al., 2013; Iwasaki and Pillai, 2014 . In addition, inflammasome activation leads to the release of IL-1β and IL-18 Pothlichet et al., 2013; Iwasaki and Pillai, 2014 . All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 .", "All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 . Furthermore, aberrant coagulation induced by virus infection causes a hyper-inflammatory response Yang and Tang, 2016 . All these events contribute to lung tissue injury Imai et al., 2008; Davidson et al., 2014 . The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . .", "The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . . On the other hand, already colonized bacteria might enhance influenza virus virulence either by directly secreting proteases that cleave and activate HA Figure 2 Bottcher-Friebertshauser et al., 2013 or, indirectly, by activating host proteases such as plasminogen, which increases replication rates and infectivity of the virus Scheiblauer et al., 1992; Tse and Whittaker, 2015 . Potentially pathogenic bacteria, including the three species mentioned above, express an arsenal of virulence factors responsible for attachment to human host structures. Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract.", "Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract. Seasonal IAV infection can lead to an increased risk of secondary bacterial infections, i.e., pneumonia. Several experimental models can be used for studying these severe infections. Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases.", "Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases. In vivo bacterial and viral co-infections are mainly performed in mice, which does not necessarily resemble the human physiology and immune system. Thus, we suggest using the porcine model, which nearly resembles over 80% of the human immune system. MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 .", "MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 . Once the initial attachment occurs, bacterial cytotoxins including pneumolysin of pneumococci Garcia-Suarez Mdel et al., 2007; Zahlten et al., 2015 , α-hemolysin and leukocidins of S. aureus . , and Streptolysins S and O and Streptococcal pyrogenic exotoxin B of S. pyogenes Tsai et al., 1998; Gurel et al., 2013; Siemens et al., 2015 Siemens et al., , 2016 , can synergize with viral counterparts to further increase lung tissue pathology. Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 .", "Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 . As the patient begins to recover from viral infection, secondary bacterial infections might occur . due to the incomplete wound healing and exposure of host membrane components, including laminin, collagens type I and IV to classical bacterial MSCRAMMs Louria et al., 1959; Puchelle et al., 2006 . Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 .", "Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 . Respiratory viruses like IAV are able to induce suppression and killing of the resident alveolar macrophages Figure 2 . . These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections .", "These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections . . In addition, induction of interferons as a response to viral infection compromises the immune sensing of Gram-positive bacteria by neutrophils and macrophages, which would normally clear the bacteria from the lungs Figure 2 Sun and Metzger, 2008; Tian et al., 2012 . The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state .", "The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state . . The subsequent infection with Gram-positive bacteria, e.g., pneumococci, enhances the type I interferon expression, which in turn suppresses production of the CCL2 chemokine and recruitment of macrophages Nakamura Frontiers in Microbiology | FIGURE 2 | The interplay between IAV, bacteria, and the human host. The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment.", "The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment. IAV is able to induce suppression and killing of resident alveolar macrophages AM , which in turn delays viral clearance. The release of viral RNA activates different immune response pathways resulting in cytokine storm. Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages.", "Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages. After initial inflammation, the situation might worsen due to cellular infiltration of the lungs by neutrophils PMN , leading to an increased degranulation and tissue damage by effector molecules, including heparin-binding protein HBP . et al., 2011 . Another study by Shahangian et al. . revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion.", ". revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion. Other studies found that IAV exposed lungs had impaired natural killer NK cell responses in the airway to subsequent S. aureus infection . . Reduced TNFα production by NK cells was identified as a crucial upstream mechanism of depressed antimicrobial activities by alveolar macrophages Figure 2 . . It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . .", "It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . . In vitro studies on the interplay between IAV-pneumococci and human dendritic cells revealed TLR3 as a crucial sensor of viral and bacterial RNA leading to enhanced IL-12p70 production, which in turn might promote an anti-viral state by upregulation of interferons Yamamoto et al., 2004; Spelmink et al., 2016 . However, it should be noted that depending on the bacterial species the disease manifestation and underlying innate immune responses might vary Sharma-Chawla et al., 2016 . A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 .", "A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 . These processes are characterized by a general anti-inflammatory state and suppression of mechanisms involved in pathogen clearance due to increased interleukin-10 production van der Sluijs et al., 2004; Metzger and Sun, 2013 . The anti-inflammatory state suppresses the expression of pattern recognition receptors PRR on professional phagocytes leading to impaired phagocytosis and killing of microbes. These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis .", "These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis . . In co-infections, after initial inflammation in response to viral infection the situation might worsen due to bacterial invasion and enhanced cellular infiltration of the lungs by neutrophils, leading to an increased tissue damage and cytokine storm Figure 2 Conenello et al., 2007; McAuley et al., 2007 McAuley et al., , 2010 Porto and Stein, 2016 . Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 .", "Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 . Bacterial pathogens also express a variety of cytolytic toxins that can contribute to inflammation and tissue pathology. Pneumolysin, a pneumococcal pore-forming toxin with low affinity to lung epithelial cells, can damage neutrophils by utilizing P2X7 receptor . . Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs .", ". Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs . . GAS toxins, including SLO and SpeB, are capable of directly causing tissue damage and promoting pro-inflammatory states through neutrophil lysis Snall et al., 2016; Uhlmann et al., 2016 . The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 .", "The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 . Figure 2 summarizes the interplay between virus, bacteria, and host. Experimental animal models are a useful tool to study in vivo effects of different infectious agents and they represent approximately 3% of all pneumonia research published in peerreview journals . . However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 .", ". However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 . Hackam and colleagues identified 2,000 articles published between 1980 and 2006 in seven leading scientific journals that regularly publish animal studies Hackam and Redelmeier, 2006 . Seventy-six out of 2,000 were highly cited with a median citation count of 889. Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 .", "Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 . In pneumonia models, mammalians are mostly used because of their anatomical and physiological proximity to humans . . To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms.", "To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms. Rapid reproductive rate, small size, less complicated handling, the ability to reproduce and compare results with already published bacterial and viral mono-infections, detailed knowledge of genetics and immune responses, and a plethora of available reagents to study infections in mice are reasons for the use of these animals. To avoid variations in responses due to genetic diversity inbred mice strains are useful tools for studies aiming to elucidate molecular mechanisms of diseases. In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 .", "In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 . However, mouse models for bacterial and/or viral infections have several limitations. Most of the bacterial and viral species under study are human pathogens. In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 .", "In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 . IAV was shown to be essential for pneumococcal transmission from colonized mice to their naive littermates and the transmission occurred only when all mice were infected with IAV . . et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 .", ". et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 . The infection of ferrets with human seasonal IAV isolates results in an upper respiratory tract infection similar to human influenza infection Tripp and Tompkins, 2009 . In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism.", "In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism. A report by Sanford and Ramsay showed enhanced staphylococcal colonization of the upper respiratory tract in IAV infected animals as compared to non-infected, while no difference between both groups was observed in group B streptococcal infection Sanford and Ramsay, 1987 . In contrast, Smith and Mc Cullers reported lack of establishment of staphylococcal infection even when ferrets were pre-infected with IAV Smith and McCullers, 2014 . The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 .", "The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 . In recent years, the guinea pig Cavia porcellus was also used in pneumonia research. The physiology and anatomy of the guinea pig lung resembles to a certain extent the human lung and this model organism is often used in non-infectious lung diseases, including asthma and chronic obstructive pulmonary disease Canning and Chou, 2008 . In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses.", "In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses. Although viral replication can be readily detected upon intranasal inoculation in the upper respiratory tract and the lungs, guinea pigs exhibit only minor clinical symptoms Lowen et al., 2006; Gabbard et al., 2014 . However, the lung pathology of human IAV infected guinea pigs correlates with the clinical severity of human infection . . Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . .", ". Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . . Guinea pigs infected with pneumococci alone and cage-mated with non-treated contact animals transmitted the bacteria only in 7% of cases, while Sendai-virus infected, co-housed guinea pigs acquired pneumococcal infection in 83% of contacts . . Another study evaluated antibiotic efficacy in invasive pulmonary infection caused by penicillin resistant pneumococcus . . Intratracheal instillation of 3 × 10 9 CFU of S. pneumoniae induced a fatal pneumonia and bacteremia in 85% of untreated animals within 46 h . . As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents.", ". As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents. This is in parts due to the lack of species specific reagents, which is a disadvantage in using this model organism. Recently, the cotton rat Sigmodon hispidus was reported to be susceptible to IAV. Nasal and pulmonary infection in adult inbred cotton rats did not require viral adaptation . . The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days.", "The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days. Tissue pathology included damage of bronchiolar epithelium and the animals developed pneumonia which persisted for nearly 3 weeks . . In bacteriological studies rats are more frequently used. There are numerous rat models investigating the impact of diabetes . , metabolic syndromes . , cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors .", ", cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors . on development of pneumococcal, streptococcal, and staphylococcal pneumonia and lung pathology. Unfortunately, there are only few studies on bacterial and viral co-infections in rats. The first was performed by Harford et al., 1946 Harford et al., 1946 . The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . .", "The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . . Another study on human respiratory syncytial virus and S. pneumoniae revealed that rats were easily colonized with pneumococci, but viral replication after subsequent infection was strain dependent. In addition, neither pneumococci nor the virus spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates . Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents.", "Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents. Rabbits Oryctolagus cuniculus are well known for their use in studying cardiovascular diseases, antibody production, and eye research. Rabbits were also employed to study pneumonia, although only a few models are available. Typical read-out parameters include survival, leukocyte infiltration of the lungs, lung pathology, and assessment of drug concentration in serum. One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . .", "One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . . This study revealed that rabbits possess an active immunity if they have recovered from one attack of experimental pneumonia and they may subsequently resist repeated intra-tracheal dosages of pneumococci Kline and Winternitz, 1913 . In 1926 an infection by inhalation of Type I pneumococci was established in rabbits Stillman and Branch, 1926 . The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 .", "The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 . However, this infection route requires surgery and species-specific reagents are scarce. In IAV research rabbits are frequently used for antibody production and for studies on antibody kinetics following single or multiple IAV administrations Loza-Tulimowska et al., 1977 . Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp.", "Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp. was investigated revealing that nasally and orally inoculated cottontails shed relatively large quantities of viral RNA . . Notably, low viral titers were found to be sufficient to initiate viral replication in cottontails . . However, despite their susceptibility to IAV infection, rabbits are only rarely used as model for IAV pathogenesis since they offer no improvement over other established infection models. Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable.", "Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable. The species most commonly used are rhesus macaques Macaca mulatta and cynomolgus macaques Macaca fasciluraris . Although it was shown early that macaques were susceptible to IAV . , the animal models of choice remained ferrets and mice. Recently, macaques have been used to compare the pathogenesis of highly virulent 1918 pandemic IAV and the pathogenic bird flu strain H5N1 with a conventional H1N1 strain . . Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . .", ". Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . . The 1918 IAV strain induced dysregulation of the antiviral response leading to insufficient protection of the host, which in turn resulted in acute respiratory distress and a fatal outcome . . The 2009 pandemic H1N1 US isolate caused severe pathological lesions in the lungs of the macaques Itoh et al., 2009 . The three studies mentioned above used combined intratracheal delivery of high doses of virus. A recent study by Marriott et al. analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . .", "analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . . Virus replication was detected in all challenge groups, although the disease remained sub-clinical. In bacteriological studies non-human primates are rarely used. For group A streptococcal infection longitudinal transcriptome analyses were performed in experimental pharyngitis . and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a .", "and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a . Another study by Olsen and colleagues analyzed the contribution of PVL of a highly virulent USA300 S. aureus strain in respiratory infection Olsen et al., 2010b . Although the lower respiratory tract disease observed in monkey mimicked the clinical and pathological features of early mild to moderate pneumonia in humans, no involvement of PVL in lung pathology or immune cell influx of the lungs could be detected Olsen et al., 2010b . The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . .", "The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . . Pneumonia progression was monitored by clinical parameters assessment, blood chemistry, nasal swabs, and pathology of the lungs. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but did not predispose the animals to subsequent severe infection with the USA300 clone . . Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade.", "Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade. In 2013, Philipp and colleagues analyzed the carriage rate of pneumococcus in 158 colony animals. None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci.", "None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci. But, when infants were colonized with 19F strain via nasopharyngeal instillation, the colonization was induced in eight of eight infants, lasted for 2 weeks in all animals and for 7 weeks in more than 60% . . The same group tested detoxified pneumolysin dPly and pneumococcal histidine triad protein D PhtD as potential vaccine candidates to prevent pneumonia . . After immunization the rhesus macaques were challenged with a 19F pneumococcal strain.", ". After immunization the rhesus macaques were challenged with a 19F pneumococcal strain. AS02-adjuvanted PhtD-dPly vaccine protected the animals against S. pneumoniae-induced pneumonia, which was linked to the capacity i to greatly reduce bacterial load within the first week post-challenge and ii the levels of PhtD-and Ply-specific antibodies . . Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified.", "Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified. Small mammals including rodents are well known from a biological, genetic, and immunological point of view and are easy to maintain. The choice of these particular animals for infectious disease studies is often a result of a compromise between technical and financial options. However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans.", "However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans. Primates are usually legally reserved to specific topics. In this case, pigs could be an appropriate model system for studying infectious diseases including pneumonia Figure 1 . The composition and size of the porcine genome is comparable to that of humans . . In addition, human and porcine organs have many common features and functions . . The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar.", ". The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar. Furthermore, like humans, pigs possess tonsils, which are absent in mice . . A major advantage of studying infectious diseases by utilizing pigs as a host organism is that pigs have a full set of innate and adaptive immune effectors. According to whole genome sequencing results the porcine immune system resembles over 80% of the human immune system, whereas mice share less than 10% with humans . . Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 .", ". Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 . In contrast to mice and similar to humans, pigs have 50-70% of circulating polymorph nuclear cells . . In addition, all functional cytokines or orthologs involved in Th1, Th2, Th17, and Treg paradigm and corresponding immune cells have been described in pigs Murtaugh et al., 2009; Kaser et al., 2011; Kiros et al., 2011 . Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . .", "Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . . In contrast to human monocytes, which can be divided in three subclasses classical CD14 + CD16 − , nonclassical CD14 + CD16 + , and intermediate CD14 ++ CD16 + , porcine monocytes consist of four subclasses Chamorro et al., 2005; Fairbairn et al., 2013 . Like human monocytes they express adhesion molecules, such as VLA-4 and LFA-1 and costimulatory molecules, including CD80 and CD86 . . The pig has previously been used to mimic a number of human infectious diseases.", ". The pig has previously been used to mimic a number of human infectious diseases. Examples for S. aureus infections with this model organism are wound infections Svedman et al., 1989 , osteomyelitis . , and sepsis . . Intravenous inoculation of piglets with pneumococci led to bacteremia during a 5 days period and was associated with fever and septic arthritis. Intranasal inoculation of piglets led to colonization for at least six consecutive days without causing clinical signs . . In addition, research on respiratory infections of pigs by human pathogens including S. aureus . , Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years.", ", Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years. The fact that pigs and humans are infected with identical subtypes of IAV H1N1, H3N2 , and show similar clinical presentation and pathogenesis, makes pigs an ideal model organism for studies on respiratory co-infections . . Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis.", "Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis. S. suis usually inhabits mucosal surfaces of tonsils, nares, genital and alimentary tract of piglets. Once the microbial balance is disturbed, the bacteria can cause meningitis, septicemia, arthritis, and pneumonia in pigs . . Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent.", ". Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent. In general, serotype 2 is most frequently isolated from diseased pigs . . S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 .", "S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 . A recent publication by Lin and colleagues on H1N1 and S. suis co-infected piglets demonstrated the synergistic effects of both pathogens . . Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . .", ". Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . . In addition, genes associated with immune responses, inflammatory cytokine production, and apoptotic pathways were highly overexpressed in the coinfected group . . Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals.", ". Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals. Although the use of animals contributes greatly to our understanding of infectious diseases, human 3D-organotypic tissue models and ex vivo organ tissues should be considered, as they are most valuable tools to study host-pathogen interactions in a more complex setting Figure 1 . Tissue engineering approaches were originally focused on regenerative medicine Langer and Vacanti, 1993 . In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 .", "In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 . The adaptability of these tissue-engineered models to multiple pathogens suggests a great potential for studies of infectious diseases. For instance, the lung tissue model relevant for pneumonia consists of lung fibroblasts embedded in a collagen matrix with a stratified epithelial layer on top . . The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 .", ". The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 . A recent publication demonstrated a two-hit-event of lung pathology in staphylococcal necrotizing pneumonia . . While the α-toxin had direct damaging effect on the lung epithelium, PVL induced lung pathology indirectly through the lysis of neutrophils . . All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 .", ". All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 . On these terms, the use of cultured ex vivo human organ biopsies, which are rare due to ethical considerations, is an additional option to study host-pathogen interactions. This ex vivo system may overcome even the limitations of the engineered tissue. In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae .", "In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae . , and IAV Nicholls et al., 2007; Chan et al., 2009 , were performed. In the human setting, most of the work focused on tropism, severity of infections, release of inflammatory mediators, and replication rates of the microorganisms. In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage .", "In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage . . However, this is just a beginning and more investigations are needed to unravel the complexity underlying these highly invasive infections. In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm.", "In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm. Despite many advances in recent years, more knowledge on mechanisms and immunology of disease progression is needed. The synergistic mechanisms between viruses and bacteria leading to enhanced morbidity and mortality are poorly understood. In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates.", "In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates. However, all have limitations. Here, we suggest using the porcine model, which provides obvious advantages in studies of human infectious diseases and should be considered much more frequent for future studies on severe infectious diseases, including pneumonia." ]

[ "Bacterial and viral co-infections of the respiratory tract are life-threatening and present a global burden to the global community. Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes are frequent colonizers of the upper respiratory tract. Imbalances through acquisition of seasonal viruses, e.g., Influenza A virus, can lead to bacterial dissemination to the lower respiratory tract, which in turn can result in severe pneumonia. In this review, we summarize the current knowledge about bacterial and viral co-infections of the respiratory tract and focus on potential experimental models suitable for mimicking this disease. Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available.", "Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available. The knowledge gained from these studies led to important discoveries and advances in understanding these infectious diseases. Nevertheless, mouse and other infection models have limitations, especially in translation of the discoveries to humans. Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 .", "Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 . The upper respiratory tract is a natural niche for potentially pathogenic bacteria embedded in commensal communities forming the nasopharyngeal microbiome. In particular, the microbial communities of the nasopharynx . are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . .", "are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . . The composition of the nasopharyngeal microbiome is highly dynamic Biesbroek et al., 2014a,b,c and many factors, including environmental and host factors, can affect microbial colonization . . Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b .", "Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b . The dynamic microbiome composition is guaranteed through the interplay between bacterial species, other microbes, and changing environmental conditions, as well as host-bacteria interactions Blaser and Falkow, 2009 . Most of the time, the microbiome and its interplay with the human host are believed to be beneficial for both Pettigrew et al., 2008; Murphy et al., 2009 . However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues .", "However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues . . While commensal respiratory microbiota facilitated immune-support against Influenza A virus infection IAV , oral treatment with antibiotics resulted not only in a shift of bacterial composition, but also in impaired CD4 T-, CD8 T-, and B-cell immunity following infection with IAV in mice . . Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls .", ". Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls . . However, another study published in the same year contradicted these results . . Chaban and colleagues analyzed microbiomes of 65 patients from H1N1 IAV outbreak in 2009. Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . .", "Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . . In this review we discuss secondary bacterial infections of the respiratory tract after primary infection by IAV with a focus on mechanisms by which these interactions are potentially mediated, and we will provide insight into the host contribution and immunological consequences. We further focus on potential animal models suitable for mimicking asymptomatic bacterial colonization and disease progression and thus, enabling to study adaptation strategies, viral-bacterial interactions, and immune responses in these highly lethal co-infections. Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . .", "Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . . The 8-segmented genomes of influenza A viruses are characterized by a significant plasticity. Due to point mutations and re-assortment events new variants or strains with epidemic or pandemic potential emerge Neumann et al., 2009 . In addition, influenza can be transmitted between animals, including swine, birds, horses, and humans, making it a zoonotic disease . . Seasonal influenza usually resolves without consequences in healthy individuals.", ". Seasonal influenza usually resolves without consequences in healthy individuals. However, it is estimated that seasonal influenza effects 5-10% of the world's population resulting in about 250,000 to 500,000 deaths annually Tjon-Kon-Fat et al., 2016 . At greater risk to develop secondary bacterial pneumonia are individuals with comorbidities, elderly people age > 65 , pregnant women, and children under the age of one . . For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 .", ". For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 . The pandemic killed around 50 million people worldwide and remains unique in its severity compared to other big outbreaks. However, many of the findings have been reinterpreted in recent years Brundage and Shanks, 2007; Chien et al., 2009 . It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . .", "It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . . Individual studies limited to certain regions identified also other pathogens commonly colonizing the respiratory tract, including Staphylococcus aureus, group A streptococcus GAS and Haemophilus influenzae Brundage and Shanks, 2008 . During the next two pandemics H2N2 Asian Flu 1957 and H3N2 Hong Kong Flu 1968 −1969 bacterial co-infections were less likely the cause of death compared to the Spanish Flu Giles and Shuttleworth, 1957; Trotter et al., 1959 . Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 .", "Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 . In 1957-1958, S. aureus was predominantly isolated from fatal pneumonia cases Hers et al., 1957 Hers et al., , 1958 Robertson et al., 1958; Martin et al., 1959 , whereas S. pneumoniae returned as predominant cause of severe pneumonia during the Hong Kong Flu Sharrar, 1969; Bisno et al., 1971; Burk et al., 1971; Schwarzmann et al., 1971 . Forty years later in 2009, a novel H1N1 virus of swine origin emerged and caused again a pandemic Dawood et al., 2009 Dawood et al., , 2012 . In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 .", "In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 . Although regional variations occurred, pneumococci and S. aureus were the most frequently isolated bacterial species Mauad et al., 2010; Shieh et al., 2010; Rice et al., 2012 . Group A streptococcus was absent in many local pneumonia outbreaks associated with viruses, but was predominant in others Brundage and Shanks, 2008; Ampofo et al., 2010 . When it does appear, it is typically third in incidence . .", "When it does appear, it is typically third in incidence . . Overall, data on pandemic outbreaks suggest that disease severity and mortality can be linked to secondary bacterial pathogens with variations depending on regions and state of immunity of the population Brundage and Shanks, 2008; Shanks et al., 2010 Shanks et al., , 2011 McCullers, 2013 . There is increasing evidence that the nasopharyngeal microbiota plays an important role in the pathogenesis of acute viral respiratory infections Teo et al., 2015; de Steenhuijsen Piters et al., 2016; Rosas-Salazar et al., 2016a,b . Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens.", "Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens. All of them are frequent colonizers of the human nasopharynx and they share many features including pathogenic mechanisms and clinical aspects Figure 1 . However, they also have unique properties. Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia.", "Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia. In the last decades, the pathogen became resistant to an increasing number of antibiotics and methicillin-resistant S. aureus MRSA is now a major cause of hospital acquired infections Hartman and Tomasz, 1984; Ubukata et al., 1989; Zetola et al., 2005 . Also the rise of community-acquired S. aureus strains is of special concern, because certain clones are associated with very severe infections . . Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 .", ". Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 . The pneumococcus is a typical colonizer of the human nasopharynx. About 20-50% of healthy children and 8-30% of healthy adults are asymptomatically colonized McCullers, 2006 . Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 .", "Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 . Group A streptococci colonize the mouth and upper respiratory tract in about 2-5% of world's population Okumura and Nizet, 2014 . The most common, non-invasive and mild infections caused by GAS are tonsillitis and pharyngitis with estimated 600 million cases per year . . Listed as number nine in the list of global killers with around 500,000 deaths annually .", ". Listed as number nine in the list of global killers with around 500,000 deaths annually . , it is obvious that this pathogen can cause severe invasive infections, including pneumonia, sepsis, streptococcal toxic shock syndrome, and necrotizing skin infections Cunningham, 2000; Carapetis et al., 2005 . Although all three pathogens are able to cause highly lethal diseases, the most fatal remains the pneumococcus, estimated to cause ca. 10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . .", "10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . . Influenza A virus binds via HA to either α2,3or α2,6-linked sialic acid at the surface of epithelial cells of the upper and lower respiratory tract . . Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways.", "Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways. Binding of viral RNA to retinoic acid inducible gene 1 induces the expression of type I and III interferons and activates transcription factor NF-κB, which in turn activates the release of pro-inflammatory cytokines Durbin et al., 2013; Iwasaki and Pillai, 2014 . In addition, inflammasome activation leads to the release of IL-1β and IL-18 Pothlichet et al., 2013; Iwasaki and Pillai, 2014 . All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 .", "All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 . Furthermore, aberrant coagulation induced by virus infection causes a hyper-inflammatory response Yang and Tang, 2016 . All these events contribute to lung tissue injury Imai et al., 2008; Davidson et al., 2014 . The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . .", "The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . . On the other hand, already colonized bacteria might enhance influenza virus virulence either by directly secreting proteases that cleave and activate HA Figure 2 Bottcher-Friebertshauser et al., 2013 or, indirectly, by activating host proteases such as plasminogen, which increases replication rates and infectivity of the virus Scheiblauer et al., 1992; Tse and Whittaker, 2015 . Potentially pathogenic bacteria, including the three species mentioned above, express an arsenal of virulence factors responsible for attachment to human host structures. Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract.", "Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract. Seasonal IAV infection can lead to an increased risk of secondary bacterial infections, i.e., pneumonia. Several experimental models can be used for studying these severe infections. Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases.", "Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases. In vivo bacterial and viral co-infections are mainly performed in mice, which does not necessarily resemble the human physiology and immune system. Thus, we suggest using the porcine model, which nearly resembles over 80% of the human immune system. MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 .", "MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 . Once the initial attachment occurs, bacterial cytotoxins including pneumolysin of pneumococci Garcia-Suarez Mdel et al., 2007; Zahlten et al., 2015 , α-hemolysin and leukocidins of S. aureus . , and Streptolysins S and O and Streptococcal pyrogenic exotoxin B of S. pyogenes Tsai et al., 1998; Gurel et al., 2013; Siemens et al., 2015 Siemens et al., , 2016 , can synergize with viral counterparts to further increase lung tissue pathology. Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 .", "Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 . As the patient begins to recover from viral infection, secondary bacterial infections might occur . due to the incomplete wound healing and exposure of host membrane components, including laminin, collagens type I and IV to classical bacterial MSCRAMMs Louria et al., 1959; Puchelle et al., 2006 . Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 .", "Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 . Respiratory viruses like IAV are able to induce suppression and killing of the resident alveolar macrophages Figure 2 . . These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections .", "These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections . . In addition, induction of interferons as a response to viral infection compromises the immune sensing of Gram-positive bacteria by neutrophils and macrophages, which would normally clear the bacteria from the lungs Figure 2 Sun and Metzger, 2008; Tian et al., 2012 . The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state .", "The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state . . The subsequent infection with Gram-positive bacteria, e.g., pneumococci, enhances the type I interferon expression, which in turn suppresses production of the CCL2 chemokine and recruitment of macrophages Nakamura Frontiers in Microbiology | FIGURE 2 | The interplay between IAV, bacteria, and the human host. The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment.", "The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment. IAV is able to induce suppression and killing of resident alveolar macrophages AM , which in turn delays viral clearance. The release of viral RNA activates different immune response pathways resulting in cytokine storm. Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages.", "Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages. After initial inflammation, the situation might worsen due to cellular infiltration of the lungs by neutrophils PMN , leading to an increased degranulation and tissue damage by effector molecules, including heparin-binding protein HBP . et al., 2011 . Another study by Shahangian et al. . revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion.", ". revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion. Other studies found that IAV exposed lungs had impaired natural killer NK cell responses in the airway to subsequent S. aureus infection . . Reduced TNFα production by NK cells was identified as a crucial upstream mechanism of depressed antimicrobial activities by alveolar macrophages Figure 2 . . It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . .", "It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . . In vitro studies on the interplay between IAV-pneumococci and human dendritic cells revealed TLR3 as a crucial sensor of viral and bacterial RNA leading to enhanced IL-12p70 production, which in turn might promote an anti-viral state by upregulation of interferons Yamamoto et al., 2004; Spelmink et al., 2016 . However, it should be noted that depending on the bacterial species the disease manifestation and underlying innate immune responses might vary Sharma-Chawla et al., 2016 . A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 .", "A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 . These processes are characterized by a general anti-inflammatory state and suppression of mechanisms involved in pathogen clearance due to increased interleukin-10 production van der Sluijs et al., 2004; Metzger and Sun, 2013 . The anti-inflammatory state suppresses the expression of pattern recognition receptors PRR on professional phagocytes leading to impaired phagocytosis and killing of microbes. These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis .", "These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis . . In co-infections, after initial inflammation in response to viral infection the situation might worsen due to bacterial invasion and enhanced cellular infiltration of the lungs by neutrophils, leading to an increased tissue damage and cytokine storm Figure 2 Conenello et al., 2007; McAuley et al., 2007 McAuley et al., , 2010 Porto and Stein, 2016 . Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 .", "Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 . Bacterial pathogens also express a variety of cytolytic toxins that can contribute to inflammation and tissue pathology. Pneumolysin, a pneumococcal pore-forming toxin with low affinity to lung epithelial cells, can damage neutrophils by utilizing P2X7 receptor . . Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs .", ". Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs . . GAS toxins, including SLO and SpeB, are capable of directly causing tissue damage and promoting pro-inflammatory states through neutrophil lysis Snall et al., 2016; Uhlmann et al., 2016 . The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 .", "The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 . Figure 2 summarizes the interplay between virus, bacteria, and host. Experimental animal models are a useful tool to study in vivo effects of different infectious agents and they represent approximately 3% of all pneumonia research published in peerreview journals . . However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 .", ". However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 . Hackam and colleagues identified 2,000 articles published between 1980 and 2006 in seven leading scientific journals that regularly publish animal studies Hackam and Redelmeier, 2006 . Seventy-six out of 2,000 were highly cited with a median citation count of 889. Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 .", "Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 . In pneumonia models, mammalians are mostly used because of their anatomical and physiological proximity to humans . . To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms.", "To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms. Rapid reproductive rate, small size, less complicated handling, the ability to reproduce and compare results with already published bacterial and viral mono-infections, detailed knowledge of genetics and immune responses, and a plethora of available reagents to study infections in mice are reasons for the use of these animals. To avoid variations in responses due to genetic diversity inbred mice strains are useful tools for studies aiming to elucidate molecular mechanisms of diseases. In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 .", "In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 . However, mouse models for bacterial and/or viral infections have several limitations. Most of the bacterial and viral species under study are human pathogens. In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 .", "In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 . IAV was shown to be essential for pneumococcal transmission from colonized mice to their naive littermates and the transmission occurred only when all mice were infected with IAV . . et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 .", ". et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 . The infection of ferrets with human seasonal IAV isolates results in an upper respiratory tract infection similar to human influenza infection Tripp and Tompkins, 2009 . In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism.", "In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism. A report by Sanford and Ramsay showed enhanced staphylococcal colonization of the upper respiratory tract in IAV infected animals as compared to non-infected, while no difference between both groups was observed in group B streptococcal infection Sanford and Ramsay, 1987 . In contrast, Smith and Mc Cullers reported lack of establishment of staphylococcal infection even when ferrets were pre-infected with IAV Smith and McCullers, 2014 . The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 .", "The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 . In recent years, the guinea pig Cavia porcellus was also used in pneumonia research. The physiology and anatomy of the guinea pig lung resembles to a certain extent the human lung and this model organism is often used in non-infectious lung diseases, including asthma and chronic obstructive pulmonary disease Canning and Chou, 2008 . In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses.", "In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses. Although viral replication can be readily detected upon intranasal inoculation in the upper respiratory tract and the lungs, guinea pigs exhibit only minor clinical symptoms Lowen et al., 2006; Gabbard et al., 2014 . However, the lung pathology of human IAV infected guinea pigs correlates with the clinical severity of human infection . . Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . .", ". Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . . Guinea pigs infected with pneumococci alone and cage-mated with non-treated contact animals transmitted the bacteria only in 7% of cases, while Sendai-virus infected, co-housed guinea pigs acquired pneumococcal infection in 83% of contacts . . Another study evaluated antibiotic efficacy in invasive pulmonary infection caused by penicillin resistant pneumococcus . . Intratracheal instillation of 3 × 10 9 CFU of S. pneumoniae induced a fatal pneumonia and bacteremia in 85% of untreated animals within 46 h . . As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents.", ". As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents. This is in parts due to the lack of species specific reagents, which is a disadvantage in using this model organism. Recently, the cotton rat Sigmodon hispidus was reported to be susceptible to IAV. Nasal and pulmonary infection in adult inbred cotton rats did not require viral adaptation . . The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days.", "The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days. Tissue pathology included damage of bronchiolar epithelium and the animals developed pneumonia which persisted for nearly 3 weeks . . In bacteriological studies rats are more frequently used. There are numerous rat models investigating the impact of diabetes . , metabolic syndromes . , cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors .", ", cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors . on development of pneumococcal, streptococcal, and staphylococcal pneumonia and lung pathology. Unfortunately, there are only few studies on bacterial and viral co-infections in rats. The first was performed by Harford et al., 1946 Harford et al., 1946 . The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . .", "The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . . Another study on human respiratory syncytial virus and S. pneumoniae revealed that rats were easily colonized with pneumococci, but viral replication after subsequent infection was strain dependent. In addition, neither pneumococci nor the virus spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates . Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents.", "Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents. Rabbits Oryctolagus cuniculus are well known for their use in studying cardiovascular diseases, antibody production, and eye research. Rabbits were also employed to study pneumonia, although only a few models are available. Typical read-out parameters include survival, leukocyte infiltration of the lungs, lung pathology, and assessment of drug concentration in serum. One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . .", "One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . . This study revealed that rabbits possess an active immunity if they have recovered from one attack of experimental pneumonia and they may subsequently resist repeated intra-tracheal dosages of pneumococci Kline and Winternitz, 1913 . In 1926 an infection by inhalation of Type I pneumococci was established in rabbits Stillman and Branch, 1926 . The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 .", "The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 . However, this infection route requires surgery and species-specific reagents are scarce. In IAV research rabbits are frequently used for antibody production and for studies on antibody kinetics following single or multiple IAV administrations Loza-Tulimowska et al., 1977 . Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp.", "Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp. was investigated revealing that nasally and orally inoculated cottontails shed relatively large quantities of viral RNA . . Notably, low viral titers were found to be sufficient to initiate viral replication in cottontails . . However, despite their susceptibility to IAV infection, rabbits are only rarely used as model for IAV pathogenesis since they offer no improvement over other established infection models. Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable.", "Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable. The species most commonly used are rhesus macaques Macaca mulatta and cynomolgus macaques Macaca fasciluraris . Although it was shown early that macaques were susceptible to IAV . , the animal models of choice remained ferrets and mice. Recently, macaques have been used to compare the pathogenesis of highly virulent 1918 pandemic IAV and the pathogenic bird flu strain H5N1 with a conventional H1N1 strain . . Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . .", ". Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . . The 1918 IAV strain induced dysregulation of the antiviral response leading to insufficient protection of the host, which in turn resulted in acute respiratory distress and a fatal outcome . . The 2009 pandemic H1N1 US isolate caused severe pathological lesions in the lungs of the macaques Itoh et al., 2009 . The three studies mentioned above used combined intratracheal delivery of high doses of virus. A recent study by Marriott et al. analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . .", "analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . . Virus replication was detected in all challenge groups, although the disease remained sub-clinical. In bacteriological studies non-human primates are rarely used. For group A streptococcal infection longitudinal transcriptome analyses were performed in experimental pharyngitis . and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a .", "and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a . Another study by Olsen and colleagues analyzed the contribution of PVL of a highly virulent USA300 S. aureus strain in respiratory infection Olsen et al., 2010b . Although the lower respiratory tract disease observed in monkey mimicked the clinical and pathological features of early mild to moderate pneumonia in humans, no involvement of PVL in lung pathology or immune cell influx of the lungs could be detected Olsen et al., 2010b . The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . .", "The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . . Pneumonia progression was monitored by clinical parameters assessment, blood chemistry, nasal swabs, and pathology of the lungs. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but did not predispose the animals to subsequent severe infection with the USA300 clone . . Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade.", "Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade. In 2013, Philipp and colleagues analyzed the carriage rate of pneumococcus in 158 colony animals. None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci.", "None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci. But, when infants were colonized with 19F strain via nasopharyngeal instillation, the colonization was induced in eight of eight infants, lasted for 2 weeks in all animals and for 7 weeks in more than 60% . . The same group tested detoxified pneumolysin dPly and pneumococcal histidine triad protein D PhtD as potential vaccine candidates to prevent pneumonia . . After immunization the rhesus macaques were challenged with a 19F pneumococcal strain.", ". After immunization the rhesus macaques were challenged with a 19F pneumococcal strain. AS02-adjuvanted PhtD-dPly vaccine protected the animals against S. pneumoniae-induced pneumonia, which was linked to the capacity i to greatly reduce bacterial load within the first week post-challenge and ii the levels of PhtD-and Ply-specific antibodies . . Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified.", "Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified. Small mammals including rodents are well known from a biological, genetic, and immunological point of view and are easy to maintain. The choice of these particular animals for infectious disease studies is often a result of a compromise between technical and financial options. However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans.", "However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans. Primates are usually legally reserved to specific topics. In this case, pigs could be an appropriate model system for studying infectious diseases including pneumonia Figure 1 . The composition and size of the porcine genome is comparable to that of humans . . In addition, human and porcine organs have many common features and functions . . The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar.", ". The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar. Furthermore, like humans, pigs possess tonsils, which are absent in mice . . A major advantage of studying infectious diseases by utilizing pigs as a host organism is that pigs have a full set of innate and adaptive immune effectors. According to whole genome sequencing results the porcine immune system resembles over 80% of the human immune system, whereas mice share less than 10% with humans . . Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 .", ". Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 . In contrast to mice and similar to humans, pigs have 50-70% of circulating polymorph nuclear cells . . In addition, all functional cytokines or orthologs involved in Th1, Th2, Th17, and Treg paradigm and corresponding immune cells have been described in pigs Murtaugh et al., 2009; Kaser et al., 2011; Kiros et al., 2011 . Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . .", "Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . . In contrast to human monocytes, which can be divided in three subclasses classical CD14 + CD16 − , nonclassical CD14 + CD16 + , and intermediate CD14 ++ CD16 + , porcine monocytes consist of four subclasses Chamorro et al., 2005; Fairbairn et al., 2013 . Like human monocytes they express adhesion molecules, such as VLA-4 and LFA-1 and costimulatory molecules, including CD80 and CD86 . . The pig has previously been used to mimic a number of human infectious diseases.", ". The pig has previously been used to mimic a number of human infectious diseases. Examples for S. aureus infections with this model organism are wound infections Svedman et al., 1989 , osteomyelitis . , and sepsis . . Intravenous inoculation of piglets with pneumococci led to bacteremia during a 5 days period and was associated with fever and septic arthritis. Intranasal inoculation of piglets led to colonization for at least six consecutive days without causing clinical signs . . In addition, research on respiratory infections of pigs by human pathogens including S. aureus . , Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years.", ", Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years. The fact that pigs and humans are infected with identical subtypes of IAV H1N1, H3N2 , and show similar clinical presentation and pathogenesis, makes pigs an ideal model organism for studies on respiratory co-infections . . Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis.", "Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis. S. suis usually inhabits mucosal surfaces of tonsils, nares, genital and alimentary tract of piglets. Once the microbial balance is disturbed, the bacteria can cause meningitis, septicemia, arthritis, and pneumonia in pigs . . Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent.", ". Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent. In general, serotype 2 is most frequently isolated from diseased pigs . . S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 .", "S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 . A recent publication by Lin and colleagues on H1N1 and S. suis co-infected piglets demonstrated the synergistic effects of both pathogens . . Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . .", ". Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . . In addition, genes associated with immune responses, inflammatory cytokine production, and apoptotic pathways were highly overexpressed in the coinfected group . . Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals.", ". Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals. Although the use of animals contributes greatly to our understanding of infectious diseases, human 3D-organotypic tissue models and ex vivo organ tissues should be considered, as they are most valuable tools to study host-pathogen interactions in a more complex setting Figure 1 . Tissue engineering approaches were originally focused on regenerative medicine Langer and Vacanti, 1993 . In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 .", "In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 . The adaptability of these tissue-engineered models to multiple pathogens suggests a great potential for studies of infectious diseases. For instance, the lung tissue model relevant for pneumonia consists of lung fibroblasts embedded in a collagen matrix with a stratified epithelial layer on top . . The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 .", ". The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 . A recent publication demonstrated a two-hit-event of lung pathology in staphylococcal necrotizing pneumonia . . While the α-toxin had direct damaging effect on the lung epithelium, PVL induced lung pathology indirectly through the lysis of neutrophils . . All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 .", ". All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 . On these terms, the use of cultured ex vivo human organ biopsies, which are rare due to ethical considerations, is an additional option to study host-pathogen interactions. This ex vivo system may overcome even the limitations of the engineered tissue. In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae .", "In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae . , and IAV Nicholls et al., 2007; Chan et al., 2009 , were performed. In the human setting, most of the work focused on tropism, severity of infections, release of inflammatory mediators, and replication rates of the microorganisms. In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage .", "In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage . . However, this is just a beginning and more investigations are needed to unravel the complexity underlying these highly invasive infections. In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm.", "In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm. Despite many advances in recent years, more knowledge on mechanisms and immunology of disease progression is needed. The synergistic mechanisms between viruses and bacteria leading to enhanced morbidity and mortality are poorly understood. In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates.", "In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates. However, all have limitations. Here, we suggest using the porcine model, which provides obvious advantages in studies of human infectious diseases and should be considered much more frequent for future studies on severe infectious diseases, including pneumonia." ]

[ "Bacterial and viral co-infections of the respiratory tract are life-threatening and present a global burden to the global community. Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes are frequent colonizers of the upper respiratory tract. Imbalances through acquisition of seasonal viruses, e.g., Influenza A virus, can lead to bacterial dissemination to the lower respiratory tract, which in turn can result in severe pneumonia. In this review, we summarize the current knowledge about bacterial and viral co-infections of the respiratory tract and focus on potential experimental models suitable for mimicking this disease. Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available.", "Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available. The knowledge gained from these studies led to important discoveries and advances in understanding these infectious diseases. Nevertheless, mouse and other infection models have limitations, especially in translation of the discoveries to humans. Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 .", "Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 . The upper respiratory tract is a natural niche for potentially pathogenic bacteria embedded in commensal communities forming the nasopharyngeal microbiome. In particular, the microbial communities of the nasopharynx . are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . .", "are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . . The composition of the nasopharyngeal microbiome is highly dynamic Biesbroek et al., 2014a,b,c and many factors, including environmental and host factors, can affect microbial colonization . . Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b .", "Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b . The dynamic microbiome composition is guaranteed through the interplay between bacterial species, other microbes, and changing environmental conditions, as well as host-bacteria interactions Blaser and Falkow, 2009 . Most of the time, the microbiome and its interplay with the human host are believed to be beneficial for both Pettigrew et al., 2008; Murphy et al., 2009 . However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues .", "However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues . . While commensal respiratory microbiota facilitated immune-support against Influenza A virus infection IAV , oral treatment with antibiotics resulted not only in a shift of bacterial composition, but also in impaired CD4 T-, CD8 T-, and B-cell immunity following infection with IAV in mice . . Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls .", ". Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls . . However, another study published in the same year contradicted these results . . Chaban and colleagues analyzed microbiomes of 65 patients from H1N1 IAV outbreak in 2009. Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . .", "Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . . In this review we discuss secondary bacterial infections of the respiratory tract after primary infection by IAV with a focus on mechanisms by which these interactions are potentially mediated, and we will provide insight into the host contribution and immunological consequences. We further focus on potential animal models suitable for mimicking asymptomatic bacterial colonization and disease progression and thus, enabling to study adaptation strategies, viral-bacterial interactions, and immune responses in these highly lethal co-infections. Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . .", "Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . . The 8-segmented genomes of influenza A viruses are characterized by a significant plasticity. Due to point mutations and re-assortment events new variants or strains with epidemic or pandemic potential emerge Neumann et al., 2009 . In addition, influenza can be transmitted between animals, including swine, birds, horses, and humans, making it a zoonotic disease . . Seasonal influenza usually resolves without consequences in healthy individuals.", ". Seasonal influenza usually resolves without consequences in healthy individuals. However, it is estimated that seasonal influenza effects 5-10% of the world's population resulting in about 250,000 to 500,000 deaths annually Tjon-Kon-Fat et al., 2016 . At greater risk to develop secondary bacterial pneumonia are individuals with comorbidities, elderly people age > 65 , pregnant women, and children under the age of one . . For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 .", ". For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 . The pandemic killed around 50 million people worldwide and remains unique in its severity compared to other big outbreaks. However, many of the findings have been reinterpreted in recent years Brundage and Shanks, 2007; Chien et al., 2009 . It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . .", "It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . . Individual studies limited to certain regions identified also other pathogens commonly colonizing the respiratory tract, including Staphylococcus aureus, group A streptococcus GAS and Haemophilus influenzae Brundage and Shanks, 2008 . During the next two pandemics H2N2 Asian Flu 1957 and H3N2 Hong Kong Flu 1968 −1969 bacterial co-infections were less likely the cause of death compared to the Spanish Flu Giles and Shuttleworth, 1957; Trotter et al., 1959 . Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 .", "Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 . In 1957-1958, S. aureus was predominantly isolated from fatal pneumonia cases Hers et al., 1957 Hers et al., , 1958 Robertson et al., 1958; Martin et al., 1959 , whereas S. pneumoniae returned as predominant cause of severe pneumonia during the Hong Kong Flu Sharrar, 1969; Bisno et al., 1971; Burk et al., 1971; Schwarzmann et al., 1971 . Forty years later in 2009, a novel H1N1 virus of swine origin emerged and caused again a pandemic Dawood et al., 2009 Dawood et al., , 2012 . In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 .", "In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 . Although regional variations occurred, pneumococci and S. aureus were the most frequently isolated bacterial species Mauad et al., 2010; Shieh et al., 2010; Rice et al., 2012 . Group A streptococcus was absent in many local pneumonia outbreaks associated with viruses, but was predominant in others Brundage and Shanks, 2008; Ampofo et al., 2010 . When it does appear, it is typically third in incidence . .", "When it does appear, it is typically third in incidence . . Overall, data on pandemic outbreaks suggest that disease severity and mortality can be linked to secondary bacterial pathogens with variations depending on regions and state of immunity of the population Brundage and Shanks, 2008; Shanks et al., 2010 Shanks et al., , 2011 McCullers, 2013 . There is increasing evidence that the nasopharyngeal microbiota plays an important role in the pathogenesis of acute viral respiratory infections Teo et al., 2015; de Steenhuijsen Piters et al., 2016; Rosas-Salazar et al., 2016a,b . Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens.", "Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens. All of them are frequent colonizers of the human nasopharynx and they share many features including pathogenic mechanisms and clinical aspects Figure 1 . However, they also have unique properties. Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia.", "Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia. In the last decades, the pathogen became resistant to an increasing number of antibiotics and methicillin-resistant S. aureus MRSA is now a major cause of hospital acquired infections Hartman and Tomasz, 1984; Ubukata et al., 1989; Zetola et al., 2005 . Also the rise of community-acquired S. aureus strains is of special concern, because certain clones are associated with very severe infections . . Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 .", ". Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 . The pneumococcus is a typical colonizer of the human nasopharynx. About 20-50% of healthy children and 8-30% of healthy adults are asymptomatically colonized McCullers, 2006 . Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 .", "Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 . Group A streptococci colonize the mouth and upper respiratory tract in about 2-5% of world's population Okumura and Nizet, 2014 . The most common, non-invasive and mild infections caused by GAS are tonsillitis and pharyngitis with estimated 600 million cases per year . . Listed as number nine in the list of global killers with around 500,000 deaths annually .", ". Listed as number nine in the list of global killers with around 500,000 deaths annually . , it is obvious that this pathogen can cause severe invasive infections, including pneumonia, sepsis, streptococcal toxic shock syndrome, and necrotizing skin infections Cunningham, 2000; Carapetis et al., 2005 . Although all three pathogens are able to cause highly lethal diseases, the most fatal remains the pneumococcus, estimated to cause ca. 10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . .", "10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . . Influenza A virus binds via HA to either α2,3or α2,6-linked sialic acid at the surface of epithelial cells of the upper and lower respiratory tract . . Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways.", "Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways. Binding of viral RNA to retinoic acid inducible gene 1 induces the expression of type I and III interferons and activates transcription factor NF-κB, which in turn activates the release of pro-inflammatory cytokines Durbin et al., 2013; Iwasaki and Pillai, 2014 . In addition, inflammasome activation leads to the release of IL-1β and IL-18 Pothlichet et al., 2013; Iwasaki and Pillai, 2014 . All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 .", "All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 . Furthermore, aberrant coagulation induced by virus infection causes a hyper-inflammatory response Yang and Tang, 2016 . All these events contribute to lung tissue injury Imai et al., 2008; Davidson et al., 2014 . The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . .", "The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . . On the other hand, already colonized bacteria might enhance influenza virus virulence either by directly secreting proteases that cleave and activate HA Figure 2 Bottcher-Friebertshauser et al., 2013 or, indirectly, by activating host proteases such as plasminogen, which increases replication rates and infectivity of the virus Scheiblauer et al., 1992; Tse and Whittaker, 2015 . Potentially pathogenic bacteria, including the three species mentioned above, express an arsenal of virulence factors responsible for attachment to human host structures. Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract.", "Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract. Seasonal IAV infection can lead to an increased risk of secondary bacterial infections, i.e., pneumonia. Several experimental models can be used for studying these severe infections. Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases.", "Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases. In vivo bacterial and viral co-infections are mainly performed in mice, which does not necessarily resemble the human physiology and immune system. Thus, we suggest using the porcine model, which nearly resembles over 80% of the human immune system. MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 .", "MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 . Once the initial attachment occurs, bacterial cytotoxins including pneumolysin of pneumococci Garcia-Suarez Mdel et al., 2007; Zahlten et al., 2015 , α-hemolysin and leukocidins of S. aureus . , and Streptolysins S and O and Streptococcal pyrogenic exotoxin B of S. pyogenes Tsai et al., 1998; Gurel et al., 2013; Siemens et al., 2015 Siemens et al., , 2016 , can synergize with viral counterparts to further increase lung tissue pathology. Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 .", "Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 . As the patient begins to recover from viral infection, secondary bacterial infections might occur . due to the incomplete wound healing and exposure of host membrane components, including laminin, collagens type I and IV to classical bacterial MSCRAMMs Louria et al., 1959; Puchelle et al., 2006 . Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 .", "Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 . Respiratory viruses like IAV are able to induce suppression and killing of the resident alveolar macrophages Figure 2 . . These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections .", "These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections . . In addition, induction of interferons as a response to viral infection compromises the immune sensing of Gram-positive bacteria by neutrophils and macrophages, which would normally clear the bacteria from the lungs Figure 2 Sun and Metzger, 2008; Tian et al., 2012 . The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state .", "The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state . . The subsequent infection with Gram-positive bacteria, e.g., pneumococci, enhances the type I interferon expression, which in turn suppresses production of the CCL2 chemokine and recruitment of macrophages Nakamura Frontiers in Microbiology | FIGURE 2 | The interplay between IAV, bacteria, and the human host. The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment.", "The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment. IAV is able to induce suppression and killing of resident alveolar macrophages AM , which in turn delays viral clearance. The release of viral RNA activates different immune response pathways resulting in cytokine storm. Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages.", "Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages. After initial inflammation, the situation might worsen due to cellular infiltration of the lungs by neutrophils PMN , leading to an increased degranulation and tissue damage by effector molecules, including heparin-binding protein HBP . et al., 2011 . Another study by Shahangian et al. . revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion.", ". revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion. Other studies found that IAV exposed lungs had impaired natural killer NK cell responses in the airway to subsequent S. aureus infection . . Reduced TNFα production by NK cells was identified as a crucial upstream mechanism of depressed antimicrobial activities by alveolar macrophages Figure 2 . . It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . .", "It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . . In vitro studies on the interplay between IAV-pneumococci and human dendritic cells revealed TLR3 as a crucial sensor of viral and bacterial RNA leading to enhanced IL-12p70 production, which in turn might promote an anti-viral state by upregulation of interferons Yamamoto et al., 2004; Spelmink et al., 2016 . However, it should be noted that depending on the bacterial species the disease manifestation and underlying innate immune responses might vary Sharma-Chawla et al., 2016 . A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 .", "A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 . These processes are characterized by a general anti-inflammatory state and suppression of mechanisms involved in pathogen clearance due to increased interleukin-10 production van der Sluijs et al., 2004; Metzger and Sun, 2013 . The anti-inflammatory state suppresses the expression of pattern recognition receptors PRR on professional phagocytes leading to impaired phagocytosis and killing of microbes. These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis .", "These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis . . In co-infections, after initial inflammation in response to viral infection the situation might worsen due to bacterial invasion and enhanced cellular infiltration of the lungs by neutrophils, leading to an increased tissue damage and cytokine storm Figure 2 Conenello et al., 2007; McAuley et al., 2007 McAuley et al., , 2010 Porto and Stein, 2016 . Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 .", "Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 . Bacterial pathogens also express a variety of cytolytic toxins that can contribute to inflammation and tissue pathology. Pneumolysin, a pneumococcal pore-forming toxin with low affinity to lung epithelial cells, can damage neutrophils by utilizing P2X7 receptor . . Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs .", ". Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs . . GAS toxins, including SLO and SpeB, are capable of directly causing tissue damage and promoting pro-inflammatory states through neutrophil lysis Snall et al., 2016; Uhlmann et al., 2016 . The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 .", "The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 . Figure 2 summarizes the interplay between virus, bacteria, and host. Experimental animal models are a useful tool to study in vivo effects of different infectious agents and they represent approximately 3% of all pneumonia research published in peerreview journals . . However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 .", ". However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 . Hackam and colleagues identified 2,000 articles published between 1980 and 2006 in seven leading scientific journals that regularly publish animal studies Hackam and Redelmeier, 2006 . Seventy-six out of 2,000 were highly cited with a median citation count of 889. Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 .", "Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 . In pneumonia models, mammalians are mostly used because of their anatomical and physiological proximity to humans . . To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms.", "To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms. Rapid reproductive rate, small size, less complicated handling, the ability to reproduce and compare results with already published bacterial and viral mono-infections, detailed knowledge of genetics and immune responses, and a plethora of available reagents to study infections in mice are reasons for the use of these animals. To avoid variations in responses due to genetic diversity inbred mice strains are useful tools for studies aiming to elucidate molecular mechanisms of diseases. In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 .", "In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 . However, mouse models for bacterial and/or viral infections have several limitations. Most of the bacterial and viral species under study are human pathogens. In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 .", "In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 . IAV was shown to be essential for pneumococcal transmission from colonized mice to their naive littermates and the transmission occurred only when all mice were infected with IAV . . et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 .", ". et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 . The infection of ferrets with human seasonal IAV isolates results in an upper respiratory tract infection similar to human influenza infection Tripp and Tompkins, 2009 . In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism.", "In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism. A report by Sanford and Ramsay showed enhanced staphylococcal colonization of the upper respiratory tract in IAV infected animals as compared to non-infected, while no difference between both groups was observed in group B streptococcal infection Sanford and Ramsay, 1987 . In contrast, Smith and Mc Cullers reported lack of establishment of staphylococcal infection even when ferrets were pre-infected with IAV Smith and McCullers, 2014 . The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 .", "The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 . In recent years, the guinea pig Cavia porcellus was also used in pneumonia research. The physiology and anatomy of the guinea pig lung resembles to a certain extent the human lung and this model organism is often used in non-infectious lung diseases, including asthma and chronic obstructive pulmonary disease Canning and Chou, 2008 . In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses.", "In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses. Although viral replication can be readily detected upon intranasal inoculation in the upper respiratory tract and the lungs, guinea pigs exhibit only minor clinical symptoms Lowen et al., 2006; Gabbard et al., 2014 . However, the lung pathology of human IAV infected guinea pigs correlates with the clinical severity of human infection . . Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . .", ". Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . . Guinea pigs infected with pneumococci alone and cage-mated with non-treated contact animals transmitted the bacteria only in 7% of cases, while Sendai-virus infected, co-housed guinea pigs acquired pneumococcal infection in 83% of contacts . . Another study evaluated antibiotic efficacy in invasive pulmonary infection caused by penicillin resistant pneumococcus . . Intratracheal instillation of 3 × 10 9 CFU of S. pneumoniae induced a fatal pneumonia and bacteremia in 85% of untreated animals within 46 h . . As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents.", ". As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents. This is in parts due to the lack of species specific reagents, which is a disadvantage in using this model organism. Recently, the cotton rat Sigmodon hispidus was reported to be susceptible to IAV. Nasal and pulmonary infection in adult inbred cotton rats did not require viral adaptation . . The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days.", "The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days. Tissue pathology included damage of bronchiolar epithelium and the animals developed pneumonia which persisted for nearly 3 weeks . . In bacteriological studies rats are more frequently used. There are numerous rat models investigating the impact of diabetes . , metabolic syndromes . , cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors .", ", cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors . on development of pneumococcal, streptococcal, and staphylococcal pneumonia and lung pathology. Unfortunately, there are only few studies on bacterial and viral co-infections in rats. The first was performed by Harford et al., 1946 Harford et al., 1946 . The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . .", "The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . . Another study on human respiratory syncytial virus and S. pneumoniae revealed that rats were easily colonized with pneumococci, but viral replication after subsequent infection was strain dependent. In addition, neither pneumococci nor the virus spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates . Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents.", "Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents. Rabbits Oryctolagus cuniculus are well known for their use in studying cardiovascular diseases, antibody production, and eye research. Rabbits were also employed to study pneumonia, although only a few models are available. Typical read-out parameters include survival, leukocyte infiltration of the lungs, lung pathology, and assessment of drug concentration in serum. One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . .", "One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . . This study revealed that rabbits possess an active immunity if they have recovered from one attack of experimental pneumonia and they may subsequently resist repeated intra-tracheal dosages of pneumococci Kline and Winternitz, 1913 . In 1926 an infection by inhalation of Type I pneumococci was established in rabbits Stillman and Branch, 1926 . The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 .", "The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 . However, this infection route requires surgery and species-specific reagents are scarce. In IAV research rabbits are frequently used for antibody production and for studies on antibody kinetics following single or multiple IAV administrations Loza-Tulimowska et al., 1977 . Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp.", "Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp. was investigated revealing that nasally and orally inoculated cottontails shed relatively large quantities of viral RNA . . Notably, low viral titers were found to be sufficient to initiate viral replication in cottontails . . However, despite their susceptibility to IAV infection, rabbits are only rarely used as model for IAV pathogenesis since they offer no improvement over other established infection models. Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable.", "Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable. The species most commonly used are rhesus macaques Macaca mulatta and cynomolgus macaques Macaca fasciluraris . Although it was shown early that macaques were susceptible to IAV . , the animal models of choice remained ferrets and mice. Recently, macaques have been used to compare the pathogenesis of highly virulent 1918 pandemic IAV and the pathogenic bird flu strain H5N1 with a conventional H1N1 strain . . Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . .", ". Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . . The 1918 IAV strain induced dysregulation of the antiviral response leading to insufficient protection of the host, which in turn resulted in acute respiratory distress and a fatal outcome . . The 2009 pandemic H1N1 US isolate caused severe pathological lesions in the lungs of the macaques Itoh et al., 2009 . The three studies mentioned above used combined intratracheal delivery of high doses of virus. A recent study by Marriott et al. analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . .", "analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . . Virus replication was detected in all challenge groups, although the disease remained sub-clinical. In bacteriological studies non-human primates are rarely used. For group A streptococcal infection longitudinal transcriptome analyses were performed in experimental pharyngitis . and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a .", "and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a . Another study by Olsen and colleagues analyzed the contribution of PVL of a highly virulent USA300 S. aureus strain in respiratory infection Olsen et al., 2010b . Although the lower respiratory tract disease observed in monkey mimicked the clinical and pathological features of early mild to moderate pneumonia in humans, no involvement of PVL in lung pathology or immune cell influx of the lungs could be detected Olsen et al., 2010b . The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . .", "The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . . Pneumonia progression was monitored by clinical parameters assessment, blood chemistry, nasal swabs, and pathology of the lungs. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but did not predispose the animals to subsequent severe infection with the USA300 clone . . Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade.", "Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade. In 2013, Philipp and colleagues analyzed the carriage rate of pneumococcus in 158 colony animals. None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci.", "None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci. But, when infants were colonized with 19F strain via nasopharyngeal instillation, the colonization was induced in eight of eight infants, lasted for 2 weeks in all animals and for 7 weeks in more than 60% . . The same group tested detoxified pneumolysin dPly and pneumococcal histidine triad protein D PhtD as potential vaccine candidates to prevent pneumonia . . After immunization the rhesus macaques were challenged with a 19F pneumococcal strain.", ". After immunization the rhesus macaques were challenged with a 19F pneumococcal strain. AS02-adjuvanted PhtD-dPly vaccine protected the animals against S. pneumoniae-induced pneumonia, which was linked to the capacity i to greatly reduce bacterial load within the first week post-challenge and ii the levels of PhtD-and Ply-specific antibodies . . Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified.", "Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified. Small mammals including rodents are well known from a biological, genetic, and immunological point of view and are easy to maintain. The choice of these particular animals for infectious disease studies is often a result of a compromise between technical and financial options. However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans.", "However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans. Primates are usually legally reserved to specific topics. In this case, pigs could be an appropriate model system for studying infectious diseases including pneumonia Figure 1 . The composition and size of the porcine genome is comparable to that of humans . . In addition, human and porcine organs have many common features and functions . . The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar.", ". The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar. Furthermore, like humans, pigs possess tonsils, which are absent in mice . . A major advantage of studying infectious diseases by utilizing pigs as a host organism is that pigs have a full set of innate and adaptive immune effectors. According to whole genome sequencing results the porcine immune system resembles over 80% of the human immune system, whereas mice share less than 10% with humans . . Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 .", ". Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 . In contrast to mice and similar to humans, pigs have 50-70% of circulating polymorph nuclear cells . . In addition, all functional cytokines or orthologs involved in Th1, Th2, Th17, and Treg paradigm and corresponding immune cells have been described in pigs Murtaugh et al., 2009; Kaser et al., 2011; Kiros et al., 2011 . Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . .", "Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . . In contrast to human monocytes, which can be divided in three subclasses classical CD14 + CD16 − , nonclassical CD14 + CD16 + , and intermediate CD14 ++ CD16 + , porcine monocytes consist of four subclasses Chamorro et al., 2005; Fairbairn et al., 2013 . Like human monocytes they express adhesion molecules, such as VLA-4 and LFA-1 and costimulatory molecules, including CD80 and CD86 . . The pig has previously been used to mimic a number of human infectious diseases.", ". The pig has previously been used to mimic a number of human infectious diseases. Examples for S. aureus infections with this model organism are wound infections Svedman et al., 1989 , osteomyelitis . , and sepsis . . Intravenous inoculation of piglets with pneumococci led to bacteremia during a 5 days period and was associated with fever and septic arthritis. Intranasal inoculation of piglets led to colonization for at least six consecutive days without causing clinical signs . . In addition, research on respiratory infections of pigs by human pathogens including S. aureus . , Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years.", ", Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years. The fact that pigs and humans are infected with identical subtypes of IAV H1N1, H3N2 , and show similar clinical presentation and pathogenesis, makes pigs an ideal model organism for studies on respiratory co-infections . . Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis.", "Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis. S. suis usually inhabits mucosal surfaces of tonsils, nares, genital and alimentary tract of piglets. Once the microbial balance is disturbed, the bacteria can cause meningitis, septicemia, arthritis, and pneumonia in pigs . . Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent.", ". Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent. In general, serotype 2 is most frequently isolated from diseased pigs . . S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 .", "S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 . A recent publication by Lin and colleagues on H1N1 and S. suis co-infected piglets demonstrated the synergistic effects of both pathogens . . Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . .", ". Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . . In addition, genes associated with immune responses, inflammatory cytokine production, and apoptotic pathways were highly overexpressed in the coinfected group . . Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals.", ". Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals. Although the use of animals contributes greatly to our understanding of infectious diseases, human 3D-organotypic tissue models and ex vivo organ tissues should be considered, as they are most valuable tools to study host-pathogen interactions in a more complex setting Figure 1 . Tissue engineering approaches were originally focused on regenerative medicine Langer and Vacanti, 1993 . In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 .", "In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 . The adaptability of these tissue-engineered models to multiple pathogens suggests a great potential for studies of infectious diseases. For instance, the lung tissue model relevant for pneumonia consists of lung fibroblasts embedded in a collagen matrix with a stratified epithelial layer on top . . The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 .", ". The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 . A recent publication demonstrated a two-hit-event of lung pathology in staphylococcal necrotizing pneumonia . . While the α-toxin had direct damaging effect on the lung epithelium, PVL induced lung pathology indirectly through the lysis of neutrophils . . All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 .", ". All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 . On these terms, the use of cultured ex vivo human organ biopsies, which are rare due to ethical considerations, is an additional option to study host-pathogen interactions. This ex vivo system may overcome even the limitations of the engineered tissue. In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae .", "In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae . , and IAV Nicholls et al., 2007; Chan et al., 2009 , were performed. In the human setting, most of the work focused on tropism, severity of infections, release of inflammatory mediators, and replication rates of the microorganisms. In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage .", "In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage . . However, this is just a beginning and more investigations are needed to unravel the complexity underlying these highly invasive infections. In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm.", "In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm. Despite many advances in recent years, more knowledge on mechanisms and immunology of disease progression is needed. The synergistic mechanisms between viruses and bacteria leading to enhanced morbidity and mortality are poorly understood. In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates.", "In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates. However, all have limitations. Here, we suggest using the porcine model, which provides obvious advantages in studies of human infectious diseases and should be considered much more frequent for future studies on severe infectious diseases, including pneumonia." ]

[ "Bacterial and viral co-infections of the respiratory tract are life-threatening and present a global burden to the global community. Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes are frequent colonizers of the upper respiratory tract. Imbalances through acquisition of seasonal viruses, e.g., Influenza A virus, can lead to bacterial dissemination to the lower respiratory tract, which in turn can result in severe pneumonia. In this review, we summarize the current knowledge about bacterial and viral co-infections of the respiratory tract and focus on potential experimental models suitable for mimicking this disease. Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available.", "Transmission of IAV and pneumonia is mainly modeled by mouse infection. Few studies utilizing ferrets, rats, guinea pigs, rabbits, and non-human primates are also available. The knowledge gained from these studies led to important discoveries and advances in understanding these infectious diseases. Nevertheless, mouse and other infection models have limitations, especially in translation of the discoveries to humans. Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 .", "Here, we suggest the use of human engineered lung tissue, human ex vivo lung tissue, and porcine models to study respiratory co-infections, which might contribute to a greater translation of the results to humans and improve both, animal and human health. Text: In recent years the human microbiota is more and more recognized to play a crucial role in pathogenesis of many diseases Weinstock, 2012 . The upper respiratory tract is a natural niche for potentially pathogenic bacteria embedded in commensal communities forming the nasopharyngeal microbiome. In particular, the microbial communities of the nasopharynx . are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . .", "are associated with respiratory diseases, i.e., severe pneumonia, which are responsible for substantial mortality and morbidity in humans worldwide . . The composition of the nasopharyngeal microbiome is highly dynamic Biesbroek et al., 2014a,b,c and many factors, including environmental and host factors, can affect microbial colonization . . Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b .", "Recent studies on neonates have shown that the respiratory microbiota develops from initially maternally transmitted mixed flora with predominance of Streptococcus viridans species to niche-specific bacterial profiles containing mostly Staphylococcus aureus at around 1 week of age Bosch et al., 2016a . Between 2 weeks and 6 months after birth, the staphylococcal predominance declines and colonization with Streptococcus pneumoniae pneumococci as a predominant pathobiont emerges Miller et al., 2011; Bosch et al., 2016a,b . The dynamic microbiome composition is guaranteed through the interplay between bacterial species, other microbes, and changing environmental conditions, as well as host-bacteria interactions Blaser and Falkow, 2009 . Most of the time, the microbiome and its interplay with the human host are believed to be beneficial for both Pettigrew et al., 2008; Murphy et al., 2009 . However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues .", "However, imbalances in microbial composition can lead to acquisition of new viral or bacterial species and invasion of potential pathogens, which in turn can become detrimental, especially in elderly people and children with an exhausted or immature immune system Pettigrew et al., 2008; Blaser and Falkow, 2009; Murphy et al., 2009 . One particular example showing imbalances introduced by single dosage of antibiotics was demonstrated by Ichinohe and colleagues . . While commensal respiratory microbiota facilitated immune-support against Influenza A virus infection IAV , oral treatment with antibiotics resulted not only in a shift of bacterial composition, but also in impaired CD4 T-, CD8 T-, and B-cell immunity following infection with IAV in mice . . Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls .", ". Analyses of human oropharyngeal microbiomes during the 2009 H1N1 IAV pandemic revealed that at the phylum level, the abundance of Fermicutes and Proteobacteria was augmented in pneumonia patients as compared to healthy controls . . However, another study published in the same year contradicted these results . . Chaban and colleagues analyzed microbiomes of 65 patients from H1N1 IAV outbreak in 2009. Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . .", "Although the phylogenetic composition of pneumonia patients was dominated by Fermicutes, Proteobacteria, and Actinobacteria, no significant differences between the patients and healthy controls or any other variables tested, including age and gender, were observed . . In this review we discuss secondary bacterial infections of the respiratory tract after primary infection by IAV with a focus on mechanisms by which these interactions are potentially mediated, and we will provide insight into the host contribution and immunological consequences. We further focus on potential animal models suitable for mimicking asymptomatic bacterial colonization and disease progression and thus, enabling to study adaptation strategies, viral-bacterial interactions, and immune responses in these highly lethal co-infections. Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . .", "Influenza A viruses belong to the family of Orthomyxoviridae and based on the antigenicity of their haemagglutinin HA and neuraminidase NA they are classified into 16 classical HA and 9 classical NA subtypes . . The 8-segmented genomes of influenza A viruses are characterized by a significant plasticity. Due to point mutations and re-assortment events new variants or strains with epidemic or pandemic potential emerge Neumann et al., 2009 . In addition, influenza can be transmitted between animals, including swine, birds, horses, and humans, making it a zoonotic disease . . Seasonal influenza usually resolves without consequences in healthy individuals.", ". Seasonal influenza usually resolves without consequences in healthy individuals. However, it is estimated that seasonal influenza effects 5-10% of the world's population resulting in about 250,000 to 500,000 deaths annually Tjon-Kon-Fat et al., 2016 . At greater risk to develop secondary bacterial pneumonia are individuals with comorbidities, elderly people age > 65 , pregnant women, and children under the age of one . . For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 .", ". For a long time it was considered that the H1N1 strain, an avian-like H1N1 virus, directly caused most of the fatalities during the 1918-1919 pandemic Spanish Flu , often from a hemorrhagic pneumonitis rapidly progressing to acute respiratory distress syndrome and death Osterholm, 2005; Gerberding, 2006; Oxford et al., 2006 . The pandemic killed around 50 million people worldwide and remains unique in its severity compared to other big outbreaks. However, many of the findings have been reinterpreted in recent years Brundage and Shanks, 2007; Chien et al., 2009 . It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . .", "It is estimated that around 95% of all severe cases and deaths were attributed to secondary infections with bacterial pathogens, most predominantly by Streptococcus pneumoniae . . Individual studies limited to certain regions identified also other pathogens commonly colonizing the respiratory tract, including Staphylococcus aureus, group A streptococcus GAS and Haemophilus influenzae Brundage and Shanks, 2008 . During the next two pandemics H2N2 Asian Flu 1957 and H3N2 Hong Kong Flu 1968 −1969 bacterial co-infections were less likely the cause of death compared to the Spanish Flu Giles and Shuttleworth, 1957; Trotter et al., 1959 . Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 .", "Still, pneumonia accounted for about 44% of deaths during the Asian Flu Giles and Shuttleworth, 1957 . Most fatalities resulting from pneumonia occurred in individuals with chronic conditions, i.e., chronic lung diseases, rheumatic carditis, and hypertension Giles and Shuttleworth, 1957 . In 1957-1958, S. aureus was predominantly isolated from fatal pneumonia cases Hers et al., 1957 Hers et al., , 1958 Robertson et al., 1958; Martin et al., 1959 , whereas S. pneumoniae returned as predominant cause of severe pneumonia during the Hong Kong Flu Sharrar, 1969; Bisno et al., 1971; Burk et al., 1971; Schwarzmann et al., 1971 . Forty years later in 2009, a novel H1N1 virus of swine origin emerged and caused again a pandemic Dawood et al., 2009 Dawood et al., , 2012 . In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 .", "In contrast to Asian and Hong Kong Flu, mortality rates were rather low, but most deaths occurred in healthy young individuals with no underlying conditions Reichert et al., 2010; Monsalvo et al., 2011; Dawood et al., 2012 . About 25-50% of severe or fatal cases were linked to complications due to bacterial pneumonia Dominguez-Cherit et al., 2009; Estenssoro et al., 2010; Mauad et al., 2010; Shieh et al., 2010 . Although regional variations occurred, pneumococci and S. aureus were the most frequently isolated bacterial species Mauad et al., 2010; Shieh et al., 2010; Rice et al., 2012 . Group A streptococcus was absent in many local pneumonia outbreaks associated with viruses, but was predominant in others Brundage and Shanks, 2008; Ampofo et al., 2010 . When it does appear, it is typically third in incidence . .", "When it does appear, it is typically third in incidence . . Overall, data on pandemic outbreaks suggest that disease severity and mortality can be linked to secondary bacterial pathogens with variations depending on regions and state of immunity of the population Brundage and Shanks, 2008; Shanks et al., 2010 Shanks et al., , 2011 McCullers, 2013 . There is increasing evidence that the nasopharyngeal microbiota plays an important role in the pathogenesis of acute viral respiratory infections Teo et al., 2015; de Steenhuijsen Piters et al., 2016; Rosas-Salazar et al., 2016a,b . Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens.", "Respiratory viruses, including IAV, have been shown to alter bacterial adherence and colonization leading to an increased risk of secondary bacterial infections Tregoning and Schwarze, 2010 . Pneumococci, S. aureus, and GAS are important human Gram-positive pathogens. All of them are frequent colonizers of the human nasopharynx and they share many features including pathogenic mechanisms and clinical aspects Figure 1 . However, they also have unique properties. Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia.", "Staphylococcus aureus colonizes persistently about 30% of the human population and typical niches include nares, axillae, and skin Peacock et al., 2001; von Eiff et al., 2001; van Belkum et al., 2009 . They cause a variety of clinical manifestations ranging from mild skin infections to fatal necrotizing pneumonia. In the last decades, the pathogen became resistant to an increasing number of antibiotics and methicillin-resistant S. aureus MRSA is now a major cause of hospital acquired infections Hartman and Tomasz, 1984; Ubukata et al., 1989; Zetola et al., 2005 . Also the rise of community-acquired S. aureus strains is of special concern, because certain clones are associated with very severe infections . . Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 .", ". Recent prospective studies demonstrated an increase in proportion of communityacquired methicillin-sensitive S. aureus in severe pneumonia cases McCaskill et al., 2007; Sicot et al., 2013 . The pneumococcus is a typical colonizer of the human nasopharynx. About 20-50% of healthy children and 8-30% of healthy adults are asymptomatically colonized McCullers, 2006 . Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 .", "Pneumococci cause diseases ranging from mild, i.e., sinusitis, conjunctivitis, and otitis media, to more severe and potentially life-threatening infections, including communityacquired pneumonia, bacteraemia, and meningitis Bogaert et al., 2004; Valles et al., 2016 . This bacterium is associated with high morbidity and mortality rates in risk groups such as immunocompromised individuals, children, and elderly Black et al., 2010; Valles et al., 2016 . Group A streptococci colonize the mouth and upper respiratory tract in about 2-5% of world's population Okumura and Nizet, 2014 . The most common, non-invasive and mild infections caused by GAS are tonsillitis and pharyngitis with estimated 600 million cases per year . . Listed as number nine in the list of global killers with around 500,000 deaths annually .", ". Listed as number nine in the list of global killers with around 500,000 deaths annually . , it is obvious that this pathogen can cause severe invasive infections, including pneumonia, sepsis, streptococcal toxic shock syndrome, and necrotizing skin infections Cunningham, 2000; Carapetis et al., 2005 . Although all three pathogens are able to cause highly lethal diseases, the most fatal remains the pneumococcus, estimated to cause ca. 10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . .", "10% of all deaths in children below 5 years of age O'Brien et al., 2009 , in the elderly . , and in immuno-compromised individuals . . Influenza A virus binds via HA to either α2,3or α2,6-linked sialic acid at the surface of epithelial cells of the upper and lower respiratory tract . . Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways.", "Seasonal strains show usually affinity to α2,6-linked sialic acids that are expressed in the human trachea, whereas avian-like viruses preferentially bind to α2,3-linked sialic acids of alveolar type II cells Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 . The release of viral genomic RNA into the cytosol activates different immune response pathways. Binding of viral RNA to retinoic acid inducible gene 1 induces the expression of type I and III interferons and activates transcription factor NF-κB, which in turn activates the release of pro-inflammatory cytokines Durbin et al., 2013; Iwasaki and Pillai, 2014 . In addition, inflammasome activation leads to the release of IL-1β and IL-18 Pothlichet et al., 2013; Iwasaki and Pillai, 2014 . All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 .", "All these responses are supposed to promote viral clearance. However, the presence of viral proteins during infection induces also direct activation of the intrinsic or indirectly the activation of the extrinsic apoptotic pathway via production of inflammatory cytokines, resulting in apoptosis or even necrosis of the epithelium Korteweg and Gu, 2008 . Furthermore, aberrant coagulation induced by virus infection causes a hyper-inflammatory response Yang and Tang, 2016 . All these events contribute to lung tissue injury Imai et al., 2008; Davidson et al., 2014 . The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . .", "The epithelial damage due to viral replication provides a beneficial environment for initial bacterial attachment . . On the other hand, already colonized bacteria might enhance influenza virus virulence either by directly secreting proteases that cleave and activate HA Figure 2 Bottcher-Friebertshauser et al., 2013 or, indirectly, by activating host proteases such as plasminogen, which increases replication rates and infectivity of the virus Scheiblauer et al., 1992; Tse and Whittaker, 2015 . Potentially pathogenic bacteria, including the three species mentioned above, express an arsenal of virulence factors responsible for attachment to human host structures. Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract.", "Microbial surface components recognizing adhesive matrix molecules FIGURE 1 | Potential models to study bacterial and viral co-infections of the respiratory tract. S. pneumoniae, S. aureus, S. pyogenes, and S. suis are frequent colonizers of the upper respiratory tract. Seasonal IAV infection can lead to an increased risk of secondary bacterial infections, i.e., pneumonia. Several experimental models can be used for studying these severe infections. Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases.", "Patient samples, including ex vivo lung tissue are materials of choice, but they are rare due to ethical considerations. Tissue engineering approaches closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ and were proven as useful tool to study infectious diseases. In vivo bacterial and viral co-infections are mainly performed in mice, which does not necessarily resemble the human physiology and immune system. Thus, we suggest using the porcine model, which nearly resembles over 80% of the human immune system. MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 .", "MSCRAMMs , such as PspC, PspA, and PsaA in pneumococci Hammerschmidt, 2006 , SPA, FnbA, ClfA, and ClfB in S. aureus Bartlett and Hulten, 2010; Otto, 2010 , and M-protein, PrtF1, and PrtF2 in GAS Cunningham, 2000 , respectively, and socalled moon-lightning proteins expressed by all three species, e.g., GAPDH, enolase or PGK . , enable the bacteria to attach to damaged cells or molecules of the extracellular matrix, including fibronectin, fibrin, fibrinogen, and collagens, or fibrinolytic proteins like plasminogen McCullers and Rehg, 2002; Bergmann and Hammerschmidt, 2007; Linke et al., 2012; Siemens et al., 2012; Voss et al., 2012 . Once the initial attachment occurs, bacterial cytotoxins including pneumolysin of pneumococci Garcia-Suarez Mdel et al., 2007; Zahlten et al., 2015 , α-hemolysin and leukocidins of S. aureus . , and Streptolysins S and O and Streptococcal pyrogenic exotoxin B of S. pyogenes Tsai et al., 1998; Gurel et al., 2013; Siemens et al., 2015 Siemens et al., , 2016 , can synergize with viral counterparts to further increase lung tissue pathology. Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 .", "Additional potential mechanisms by which the initial colonization of the lower respiratory tract and lung tissue damage might occur include potentiation of the development of pneumonia by IAV neuraminidase through enzymatic removal of sialic acid from the lung, thus exposing host receptors for pneumococcal adherence McCullers and Bartmess, 2003 . The host inflammatory state in response to viral infection can alter presentation of receptors on the surface, thus allowing bacterial invasion Cundell and Tuomanen, 1994 . As the patient begins to recover from viral infection, secondary bacterial infections might occur . due to the incomplete wound healing and exposure of host membrane components, including laminin, collagens type I and IV to classical bacterial MSCRAMMs Louria et al., 1959; Puchelle et al., 2006 . Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 .", "Epithelial cells are the first responders to infections in the lung, followed by the tissue resident alveolar macrophages. They promote viral clearance via phagocytosis, efferocytosis, and release of cytokines and chemokines to promote immune responses Hashimoto et al., 2007; Kumagai et al., 2007; Wang et al., 2012; Hillaire et al., 2013 . Respiratory viruses like IAV are able to induce suppression and killing of the resident alveolar macrophages Figure 2 . . These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections .", "These cells are usually replaced by differentiation of recruited blood derived monocytes into macrophages of different polarization patterns. This in turn creates a delay in pathogen clearance and opens a window for host susceptibility to secondary bacterial infections, colloquially named superinfections . . In addition, induction of interferons as a response to viral infection compromises the immune sensing of Gram-positive bacteria by neutrophils and macrophages, which would normally clear the bacteria from the lungs Figure 2 Sun and Metzger, 2008; Tian et al., 2012 . The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state .", "The exact mechanism underlying this phenomenon is still not understood. Several studies suggested that viral RNA activates Toll-like receptors TLR 2 and TLR4 and, consequently, the production of type I interferons to promote an antiviral state . . The subsequent infection with Gram-positive bacteria, e.g., pneumococci, enhances the type I interferon expression, which in turn suppresses production of the CCL2 chemokine and recruitment of macrophages Nakamura Frontiers in Microbiology | FIGURE 2 | The interplay between IAV, bacteria, and the human host. The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment.", "The epithelial damage due to viral replication provides a beneficial environment for bacterial Bact. attachment. IAV is able to induce suppression and killing of resident alveolar macrophages AM , which in turn delays viral clearance. The release of viral RNA activates different immune response pathways resulting in cytokine storm. Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages.", "Type I and III interferons compromise the immune recognition of Gram-positive bacteria by neutrophils and macrophages. In addition, they might suppress natural killer cell function NK , including release of TNF, which activates alveolar macrophages. After initial inflammation, the situation might worsen due to cellular infiltration of the lungs by neutrophils PMN , leading to an increased degranulation and tissue damage by effector molecules, including heparin-binding protein HBP . et al., 2011 . Another study by Shahangian et al. . revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion.", ". revealed that the antiviral state leads to impaired production of neutrophil chemoattractants CXCL1 and CXCL2, which in turn promotes less effective immune responses due to attenuated neutrophil functions during the early phase of pneumococcal invasion. Other studies found that IAV exposed lungs had impaired natural killer NK cell responses in the airway to subsequent S. aureus infection . . Reduced TNFα production by NK cells was identified as a crucial upstream mechanism of depressed antimicrobial activities by alveolar macrophages Figure 2 . . It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . .", "It seems likely that IAV NA is also able to activate host cell receptors in a TGF-β dependent manner, which in turn promotes GAS invasion and subsequent lung pathology . . In vitro studies on the interplay between IAV-pneumococci and human dendritic cells revealed TLR3 as a crucial sensor of viral and bacterial RNA leading to enhanced IL-12p70 production, which in turn might promote an anti-viral state by upregulation of interferons Yamamoto et al., 2004; Spelmink et al., 2016 . However, it should be noted that depending on the bacterial species the disease manifestation and underlying innate immune responses might vary Sharma-Chawla et al., 2016 . A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 .", "A lot of the experimental studies on disease mechanisms and immune responses are based on a subsequent bacterial infection within hours or a few days post IAV infection. However, bacterial infiltrations of the lungs might occur much later, i.e., during the onset of wound healing after partial clearance of IAV, which has been reported in most studies performed in recent years Snelgrove et al., 2008; Hussell and Cavanagh, 2009 . These processes are characterized by a general anti-inflammatory state and suppression of mechanisms involved in pathogen clearance due to increased interleukin-10 production van der Sluijs et al., 2004; Metzger and Sun, 2013 . The anti-inflammatory state suppresses the expression of pattern recognition receptors PRR on professional phagocytes leading to impaired phagocytosis and killing of microbes. These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis .", "These events might allow bacterial overgrowth in the lungs and tissue pathology Sun and Metzger, 2008; Goulding et al., 2011 . Like other severe infectious diseases caused by single agents, pneumonia is characterized by hyper-inflammatory conditions of the lungs at the onset of infection followed by a hypoinflammatory state with immune paralysis . . In co-infections, after initial inflammation in response to viral infection the situation might worsen due to bacterial invasion and enhanced cellular infiltration of the lungs by neutrophils, leading to an increased tissue damage and cytokine storm Figure 2 Conenello et al., 2007; McAuley et al., 2007 McAuley et al., , 2010 Porto and Stein, 2016 . Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 .", "Furthermore, the coagulation system becomes activated and contributes to the pathophysiological response to infection van der Poll and Herwald, 2014 . Bacteria like pneumococci, S. aureus, and GAS can activate and modulate the coagulation system, leading to extensive expression of tissue factor and increasing the risk of severe coagulopathy Shannon et al., 2013; Walters et al., 2016 . Bacterial pathogens also express a variety of cytolytic toxins that can contribute to inflammation and tissue pathology. Pneumolysin, a pneumococcal pore-forming toxin with low affinity to lung epithelial cells, can damage neutrophils by utilizing P2X7 receptor . . Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs .", ". Staphylococcal cytotoxins α-toxin and leukocidins, including Panton-Valentine leucocidin, PVL are associated with severe tissue pathology, strong upregulation of chemokines, and increased neutrophil influx of the lungs . . GAS toxins, including SLO and SpeB, are capable of directly causing tissue damage and promoting pro-inflammatory states through neutrophil lysis Snall et al., 2016; Uhlmann et al., 2016 . The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 .", "The cytolytic effects caused by bacterial toxins might synergize with the outcome of IAV cytotoxic accessory protein, PB1-F2, mediated tissue pathology leading to enhanced cytokine production Ramos and Fernandez-Sesma, 2012 . Taken together, most likely synergistic effects of the pathways that are involved in bacterial and viral inflammation lead to enhanced immune activation and higher morbidity and mortality Joyce et al., 2009; Koppe et al., 2012; Ramos and Fernandez-Sesma, 2012; Bucasas et al., 2013; Kuri et al., 2013 . Figure 2 summarizes the interplay between virus, bacteria, and host. Experimental animal models are a useful tool to study in vivo effects of different infectious agents and they represent approximately 3% of all pneumonia research published in peerreview journals . . However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 .", ". However, the constant increase of animal studies in the last decades is in contrast to their reproducibility in humans Hackam and Redelmeier, 2006 . Hackam and colleagues identified 2,000 articles published between 1980 and 2006 in seven leading scientific journals that regularly publish animal studies Hackam and Redelmeier, 2006 . Seventy-six out of 2,000 were highly cited with a median citation count of 889. Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 .", "Out of these 76 studies 28 were replicated in human randomized trials, 14 were contradicted, and 34 remained untested Hackam and Redelmeier, 2006 . Only 1.4% of the animal studies published in high-impact journals were translated in human randomized trials Hackam and Redelmeier, 2006 , whereas about 44% replication rate was reported for highly cited human studies Ioannidis, 2005 . In pneumonia models, mammalians are mostly used because of their anatomical and physiological proximity to humans . . To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms.", "To monitor extensive physiological studies, larger mammalian species, including ferrets, dogs, rabbits, pigs, and baboons are the models of choice Mizgerd and Skerrett, 2008 . However, rodents and in particular mice are used more frequently as a pneumonia model organisms. Rapid reproductive rate, small size, less complicated handling, the ability to reproduce and compare results with already published bacterial and viral mono-infections, detailed knowledge of genetics and immune responses, and a plethora of available reagents to study infections in mice are reasons for the use of these animals. To avoid variations in responses due to genetic diversity inbred mice strains are useful tools for studies aiming to elucidate molecular mechanisms of diseases. In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 .", "In addition, genetic engineering allowed to generate a wide variety of mouse variants with gainof-function, loss-of-function or reporter genes Mizgerd and Skerrett, 2008 . As outlined above, many in vivo mice studies on bacterial and viral co-infections provided useful insights into severe pneumonia, including i the fact that viral infection primes the host for bacterial susceptibility leading to severe secondary infection Hashimoto et al., 2007; Shahangian et al., 2009; Chaussee et al., 2011; Nakamura et al., 2011 , ii pathogen synergism Tsai et al., 1998; McCullers and Rehg, 2002; Garcia-Suarez Mdel et al., 2007; Gurel et al., 2013; Mairpady Shambat et al., 2015; Zahlten et al., 2015 , iii enhanced inflammatory response at the onset of infection Korteweg and Gu, 2008; Durbin et al., 2013; Pothlichet et al., 2013; Iwasaki and Pillai, 2014 leading to increased alveolar damage followed by immune paralysis with defective clearance of microorganisms Shinya et al., 2006; van Riel et al., 2007 van Riel et al., , 2010 , and iv host receptor availability for sustained bacterial infection Louria et al., 1959; Plotkowski et al., 1993; Cundell and Tuomanen, 1994; Puchelle et al., 2006; Korteweg and Gu, 2008 . However, mouse models for bacterial and/or viral infections have several limitations. Most of the bacterial and viral species under study are human pathogens. In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 .", "In recent years it was also shown that host genetic variations and sex differences have an impact on predisposition, severity, and outcome of infection Chella Krishnan et al., 2015 While C57BL/6 and BALB/c mice are characterized by a higher resistance, DBA/2 strains are more susceptible and permissive to bacterial and viral strains Alymova et al., 2011; Chella Krishnan et al., 2015 . In addition, transmission of IAV and bacteria is inefficient in adult mice, thus requiring alternative animal models, including neonatal mice or ferrets Diavatopoulos et al., 2010; McCullers et al., 2010 . IAV was shown to be essential for pneumococcal transmission from colonized mice to their naive littermates and the transmission occurred only when all mice were infected with IAV . . et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 .", ". et al., 2010 . Ferrets are naturally susceptible to IAV isolated from different species, including humans, birds, and swine Thangavel and Bouvier, 2014 . The infection of ferrets with human seasonal IAV isolates results in an upper respiratory tract infection similar to human influenza infection Tripp and Tompkins, 2009 . In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism.", "In contrast to mice, non-adapted human IAV can be used for the infection. Unfortunately, there are only few reports on bacterial and IAV co-infections in this model organism. A report by Sanford and Ramsay showed enhanced staphylococcal colonization of the upper respiratory tract in IAV infected animals as compared to non-infected, while no difference between both groups was observed in group B streptococcal infection Sanford and Ramsay, 1987 . In contrast, Smith and Mc Cullers reported lack of establishment of staphylococcal infection even when ferrets were pre-infected with IAV Smith and McCullers, 2014 . The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 .", "The biggest advantages of using ferrets as a model include i their susceptibility to nonadapted human pathogens, ii efficiency in transmitting IAV and bacteria from one individual to another, and iii presentation of the clinical signs of disease manifestation akin to human influenza infection. Unfortunately, their limited availability, complex husbandry, and limited accessibility to ferret-specific reagents makes this research difficult to perform Bouvier and Lowen, 2010 . In recent years, the guinea pig Cavia porcellus was also used in pneumonia research. The physiology and anatomy of the guinea pig lung resembles to a certain extent the human lung and this model organism is often used in non-infectious lung diseases, including asthma and chronic obstructive pulmonary disease Canning and Chou, 2008 . In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses.", "In addition, its commercial availability, ease of husbandry, the ability to work with nonadapted pathogens and the efficiency of transmission are reasons for using this in vivo model Bouvier and Lowen, 2010 . Guinea pigs are susceptible to human, avian, and swine influenza viruses. Although viral replication can be readily detected upon intranasal inoculation in the upper respiratory tract and the lungs, guinea pigs exhibit only minor clinical symptoms Lowen et al., 2006; Gabbard et al., 2014 . However, the lung pathology of human IAV infected guinea pigs correlates with the clinical severity of human infection . . Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . .", ". Transmission of pneumococci in guinea pigs is promoted by co-infection with Sendai virus . . Guinea pigs infected with pneumococci alone and cage-mated with non-treated contact animals transmitted the bacteria only in 7% of cases, while Sendai-virus infected, co-housed guinea pigs acquired pneumococcal infection in 83% of contacts . . Another study evaluated antibiotic efficacy in invasive pulmonary infection caused by penicillin resistant pneumococcus . . Intratracheal instillation of 3 × 10 9 CFU of S. pneumoniae induced a fatal pneumonia and bacteremia in 85% of untreated animals within 46 h . . As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents.", ". As with ferrets, there is a paucity of data describing immune responses to pulmonary infectious agents. This is in parts due to the lack of species specific reagents, which is a disadvantage in using this model organism. Recently, the cotton rat Sigmodon hispidus was reported to be susceptible to IAV. Nasal and pulmonary infection in adult inbred cotton rats did not require viral adaptation . . The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days.", "The infection led to increased breathing rates accompanied by weight loss and decreased body temperature. Replication of IAV was more extensive in nasal tissues than the lung, and persisted for six consecutive days. Tissue pathology included damage of bronchiolar epithelium and the animals developed pneumonia which persisted for nearly 3 weeks . . In bacteriological studies rats are more frequently used. There are numerous rat models investigating the impact of diabetes . , metabolic syndromes . , cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors .", ", cirrhosis , pharmaco-kinetics and dynamics Antonopoulou et al., 2015; Hoover et al., 2015 , intoxication . , immunization Iinuma and Okinaga, 1989 , and general bacterial virulence factors . on development of pneumococcal, streptococcal, and staphylococcal pneumonia and lung pathology. Unfortunately, there are only few studies on bacterial and viral co-infections in rats. The first was performed by Harford et al., 1946 Harford et al., 1946 . The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . .", "The authors concluded that the secondary bacterial pneumonia does not convert the sub-lethal viral infection to a lethal outcome . . Another study on human respiratory syncytial virus and S. pneumoniae revealed that rats were easily colonized with pneumococci, but viral replication after subsequent infection was strain dependent. In addition, neither pneumococci nor the virus spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates . Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents.", "Although rats share a lot of immune features with humans, including nitric oxide production by macrophages . , the biggest disadvantages are low animal availability, aggressiveness of the species, and the lack of specific reagents. Rabbits Oryctolagus cuniculus are well known for their use in studying cardiovascular diseases, antibody production, and eye research. Rabbits were also employed to study pneumonia, although only a few models are available. Typical read-out parameters include survival, leukocyte infiltration of the lungs, lung pathology, and assessment of drug concentration in serum. One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . .", "One of the first studies on pneumococcal pneumonia in rabbits was performed in Kline and Winternitz . . This study revealed that rabbits possess an active immunity if they have recovered from one attack of experimental pneumonia and they may subsequently resist repeated intra-tracheal dosages of pneumococci Kline and Winternitz, 1913 . In 1926 an infection by inhalation of Type I pneumococci was established in rabbits Stillman and Branch, 1926 . The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 .", "The bacteria infiltrated easily the lower respiratory tract and pneumococci which reached the lungs usually disappeared within hours and fatal septicemia appeared in some of the animals Stillman and Branch, 1926 . Most recent rabbit models of pneumococcal and staphylococcal pneumonia are based on intra-bronchial or intra-pulmonary infections which make them useful for pathogenesis Diep et al., 2010 Diep et al., , 2017 , as well as drug efficiency and efficacy studies Cabellos et al., 1992; Croisier-Bertin et al., 2011 . However, this infection route requires surgery and species-specific reagents are scarce. In IAV research rabbits are frequently used for antibody production and for studies on antibody kinetics following single or multiple IAV administrations Loza-Tulimowska et al., 1977 . Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp.", "Also, rabbits are used for safety investigations of vaccines e.g., CoVaccine HT or Aflunov Heldens et al., 2010; Gasparini et al., 2012 . In recent years the shedding of avian IAV by cottontails Sylvilagus spp. was investigated revealing that nasally and orally inoculated cottontails shed relatively large quantities of viral RNA . . Notably, low viral titers were found to be sufficient to initiate viral replication in cottontails . . However, despite their susceptibility to IAV infection, rabbits are only rarely used as model for IAV pathogenesis since they offer no improvement over other established infection models. Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable.", "Macaques represent the major non-human primate for studying infectious diseases. They are omnivorous and adaptable. The species most commonly used are rhesus macaques Macaca mulatta and cynomolgus macaques Macaca fasciluraris . Although it was shown early that macaques were susceptible to IAV . , the animal models of choice remained ferrets and mice. Recently, macaques have been used to compare the pathogenesis of highly virulent 1918 pandemic IAV and the pathogenic bird flu strain H5N1 with a conventional H1N1 strain . . Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . .", ". Cynomolgus macaques infected with highly pathogenic H5N1 developed acute respiratory distress syndrome, fever, and necrotizing pneumonia . . The 1918 IAV strain induced dysregulation of the antiviral response leading to insufficient protection of the host, which in turn resulted in acute respiratory distress and a fatal outcome . . The 2009 pandemic H1N1 US isolate caused severe pathological lesions in the lungs of the macaques Itoh et al., 2009 . The three studies mentioned above used combined intratracheal delivery of high doses of virus. A recent study by Marriott et al. analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . .", "analyzed the outcome of challenge routes, including inhaled aerosol and intra-nasal instillation with low to moderate doses of H1N1 in cynomolgus macaques . . Virus replication was detected in all challenge groups, although the disease remained sub-clinical. In bacteriological studies non-human primates are rarely used. For group A streptococcal infection longitudinal transcriptome analyses were performed in experimental pharyngitis . and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a .", "and lower respiratory tract infection in cynomolgus macaques Olsen et al., 2010a . The lower respiratory tract disease observed in macaques after GAS infection mimicked the clinical and pathological features of severe bronchopneumonia in humans Olsen et al., 2010a . Another study by Olsen and colleagues analyzed the contribution of PVL of a highly virulent USA300 S. aureus strain in respiratory infection Olsen et al., 2010b . Although the lower respiratory tract disease observed in monkey mimicked the clinical and pathological features of early mild to moderate pneumonia in humans, no involvement of PVL in lung pathology or immune cell influx of the lungs could be detected Olsen et al., 2010b . The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . .", "The same research group has developed a non-lethal IAV H3N2 -S. aureus co-infection model in cynomolgus macaques . . Pneumonia progression was monitored by clinical parameters assessment, blood chemistry, nasal swabs, and pathology of the lungs. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but did not predispose the animals to subsequent severe infection with the USA300 clone . . Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade.", "Although macaques are frequently used for evaluation of pneumococcal vaccine efficacy, including testing the impact of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine on antigen-specific memory B cell repertoires . , only two studies on pneumococcal carriage and pneumonia were conducted in the last decade. In 2013, Philipp and colleagues analyzed the carriage rate of pneumococcus in 158 colony animals. None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci.", "None of the surveyed rhesus macaques carried S. pneumoniae in the nasopharynx . . The authors concluded that rhesus macaque is probably not a natural host of pneumococci. But, when infants were colonized with 19F strain via nasopharyngeal instillation, the colonization was induced in eight of eight infants, lasted for 2 weeks in all animals and for 7 weeks in more than 60% . . The same group tested detoxified pneumolysin dPly and pneumococcal histidine triad protein D PhtD as potential vaccine candidates to prevent pneumonia . . After immunization the rhesus macaques were challenged with a 19F pneumococcal strain.", ". After immunization the rhesus macaques were challenged with a 19F pneumococcal strain. AS02-adjuvanted PhtD-dPly vaccine protected the animals against S. pneumoniae-induced pneumonia, which was linked to the capacity i to greatly reduce bacterial load within the first week post-challenge and ii the levels of PhtD-and Ply-specific antibodies . . Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified.", "Although only a few macaque studies on pneumonia exist, due to the close proximity to humans in terms of physiology and immunity, these animals can be a good model in the context of translational studies evaluating therapeutics and prophylaxis. Despite the wide use of different animal models, the optimal in vivo model for human pneumonia remains to be identified. Small mammals including rodents are well known from a biological, genetic, and immunological point of view and are easy to maintain. The choice of these particular animals for infectious disease studies is often a result of a compromise between technical and financial options. However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans.", "However, they are also far from humans' anatomy, physiology, immunology, and susceptibility to exclusively human pathogens. The experimental animal model should be chosen based on responses comparable to humans. Primates are usually legally reserved to specific topics. In this case, pigs could be an appropriate model system for studying infectious diseases including pneumonia Figure 1 . The composition and size of the porcine genome is comparable to that of humans . . In addition, human and porcine organs have many common features and functions . . The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar.", ". The upper respiratory tract of humans and pigs, including the lymphoid tissue in the nasopharynx, is anatomically similar. Furthermore, like humans, pigs possess tonsils, which are absent in mice . . A major advantage of studying infectious diseases by utilizing pigs as a host organism is that pigs have a full set of innate and adaptive immune effectors. According to whole genome sequencing results the porcine immune system resembles over 80% of the human immune system, whereas mice share less than 10% with humans . . Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 .", ". Most of the immune cell compartments identified in humans are also present in pigs Piriou-Guzylack and Salmon, 2008; Fairbairn et al., 2011 . In contrast to mice and similar to humans, pigs have 50-70% of circulating polymorph nuclear cells . . In addition, all functional cytokines or orthologs involved in Th1, Th2, Th17, and Treg paradigm and corresponding immune cells have been described in pigs Murtaugh et al., 2009; Kaser et al., 2011; Kiros et al., 2011 . Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . .", "Especially the very prominent human pro-inflammatory chemo-attractant, CXCL8, is present as an ortholog in pigs, whereas there is no homologue in mice . . In contrast to human monocytes, which can be divided in three subclasses classical CD14 + CD16 − , nonclassical CD14 + CD16 + , and intermediate CD14 ++ CD16 + , porcine monocytes consist of four subclasses Chamorro et al., 2005; Fairbairn et al., 2013 . Like human monocytes they express adhesion molecules, such as VLA-4 and LFA-1 and costimulatory molecules, including CD80 and CD86 . . The pig has previously been used to mimic a number of human infectious diseases.", ". The pig has previously been used to mimic a number of human infectious diseases. Examples for S. aureus infections with this model organism are wound infections Svedman et al., 1989 , osteomyelitis . , and sepsis . . Intravenous inoculation of piglets with pneumococci led to bacteremia during a 5 days period and was associated with fever and septic arthritis. Intranasal inoculation of piglets led to colonization for at least six consecutive days without causing clinical signs . . In addition, research on respiratory infections of pigs by human pathogens including S. aureus . , Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years.", ", Mycobacterium tuberculosis . , Bordetella pertussis . , Pseudomonas aeruginosa . , and IAV . , was performed in recent years. The fact that pigs and humans are infected with identical subtypes of IAV H1N1, H3N2 , and show similar clinical presentation and pathogenesis, makes pigs an ideal model organism for studies on respiratory co-infections . . Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis.", "Especially IAV infections are already well established in swine Van Reeth et al., 1998 , 2002a Jung et al., 2007; Khatri et al., 2010; Barbe et al., 2011 . In addition to the limited number of publications on pigs and human pathogens, a lot can be translated and learned from studies on the porcine zoonotic pathogen Streptococcus suis. S. suis usually inhabits mucosal surfaces of tonsils, nares, genital and alimentary tract of piglets. Once the microbial balance is disturbed, the bacteria can cause meningitis, septicemia, arthritis, and pneumonia in pigs . . Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent.", ". Some S. suis strains are considered to be hyper-virulent and others hypo-or avirulent. In general, serotype 2 is most frequently isolated from diseased pigs . . S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 .", "S. suis can also cause severe diseases in humans including septicemia, meningitis, arthritis, and streptococcal toxic shock syndrome Tang et al., 2006; Yu et al., 2006; Gottschalk et al., 2007 . Although many in vivo studies on S. suis have been performed by utilizing mice as a model organism Seitz et al., 2012; Auger et al., 2016 , several other studies have shown the advantage of using swine as a natural host for S. suis Bi et al., 2014; Ferrando et al., 2015 . A recent publication by Lin and colleagues on H1N1 and S. suis co-infected piglets demonstrated the synergistic effects of both pathogens . . Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . .", ". Co-infected piglets had more severe clinical presentation and pathological changes in the lung, as compared to animals infected with single pathogens . . In addition, genes associated with immune responses, inflammatory cytokine production, and apoptotic pathways were highly overexpressed in the coinfected group . . Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals.", ". Although the porcine model seems to be ideal to mimic human infectious diseases, there are also disadvantages, including, e.g., requirement for specialized experimental animal facilities, time consuming management, high maintenance costs, and limited availability of transgenic animals. Although the use of animals contributes greatly to our understanding of infectious diseases, human 3D-organotypic tissue models and ex vivo organ tissues should be considered, as they are most valuable tools to study host-pathogen interactions in a more complex setting Figure 1 . Tissue engineering approaches were originally focused on regenerative medicine Langer and Vacanti, 1993 . In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 .", "In contrast to standard monolayer cell cultures, tissue models much more closely resemble the 3D architecture, cellular composition, and matrix complexity of the respective organ. In recent years tissue engineering was also successfully employed in a number of studies in infectious diseases, including Zika virus infections of cerebral organoids Lancaster et al., 2013; Dang et al., 2016 , Helicobacter pylori infections of gastric epithelial organoids McCracken et al., 2014; Schlaermann et al., 2016 , Escherichia coli and Rotavirus infections of gastrointestinal and small intestinal enteroids Saxena et al., 2015; VanDussen et al., 2015 , Entamoeba histolytica or Hepatitis B virus infections of hepatic sinusoid tissue Petropolis et al., 2014 Petropolis et al., , 2016 , group A and G streptococcal or staphylococcal infections of skin tissue models Mairpady Shambat et al., 2016 , and staphylococcal and Andes hantavirus infections of human lung tissue Mairpady Shambat et al., 2015; Sundstrom et al., 2016 . The adaptability of these tissue-engineered models to multiple pathogens suggests a great potential for studies of infectious diseases. For instance, the lung tissue model relevant for pneumonia consists of lung fibroblasts embedded in a collagen matrix with a stratified epithelial layer on top . . The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 .", ". The engineered tissue is suitable for implanting and studying immune cells, including dendritic cells, monocytes, macrophages, and even peripheral blood mononuclear cells Nguyen Hoang et al., 2012; Mairpady Shambat et al., 2015 . A recent publication demonstrated a two-hit-event of lung pathology in staphylococcal necrotizing pneumonia . . While the α-toxin had direct damaging effect on the lung epithelium, PVL induced lung pathology indirectly through the lysis of neutrophils . . All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 .", ". All the studies mentioned above highlight a significant progress in the field of infectious diseases not only from a scientific point of view but also by contributing to the three R principle of animal experimentation Russell, 1995 . On these terms, the use of cultured ex vivo human organ biopsies, which are rare due to ethical considerations, is an additional option to study host-pathogen interactions. This ex vivo system may overcome even the limitations of the engineered tissue. In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae .", "In recent years human ex vivo lung tissue infections with various microorganisms, including pneumococci Szymanski et al., 2012; Fatykhova et al., 2015 , Bacillus anthracis . , Haemophilus influenzae . , and IAV Nicholls et al., 2007; Chan et al., 2009 , were performed. In the human setting, most of the work focused on tropism, severity of infections, release of inflammatory mediators, and replication rates of the microorganisms. In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage .", "In addition, recently also experiments on swine influenza virus SIV and S. suis co-infections of the porcine ex vivo lung slices were reported. Meng and colleagues showed that SIV promotes subsequent bacterial infections in a two-step process of which the first initial step was dependent on capsule expression, whereas the second step of bacterial invasion into deeper layers was capsuleindependent and required virus-mediated damage . . However, this is just a beginning and more investigations are needed to unravel the complexity underlying these highly invasive infections. In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm.", "In summary, bacterial and viral co-infections of the respiratory tract are highly lethal and present a dramatic burden for the global health system. The synergy between bacterial and viral infectious agents is related to a variety of factors, including epithelial barrier damage, exaggerated innate immune response, and cytokine storm. Despite many advances in recent years, more knowledge on mechanisms and immunology of disease progression is needed. The synergistic mechanisms between viruses and bacteria leading to enhanced morbidity and mortality are poorly understood. In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates.", "In vivo characterizations of these severe infections are mainly performed in mice which poorly resemble the human physiology and immune system. Several efforts have been made to establish other models, including ferrets, guinea pigs, rabbits, rats, and non-human primates. However, all have limitations. Here, we suggest using the porcine model, which provides obvious advantages in studies of human infectious diseases and should be considered much more frequent for future studies on severe infectious diseases, including pneumonia." ]

[ "Inovirus-associated vectors IAVs are engineered, non-lytic, filamentous bacteriophages that are assembled primarily from thousands of copies of the major coat protein gp8 and just five copies of each of the four minor coat proteins gp3, gp6, gp7 and gp9. Inovirus display studies have shown that the architecture of inoviruses makes all coat proteins of the inoviral particle accessible to the outside. This particular feature of IAVs allows foreign antigenic peptides to be displayed on the outer surface of the virion fused to its coat proteins and for more than two decades has been exploited in many applications including antibody or peptide display libraries, drug design, and vaccine development against infectious and non-infectious diseases. As vaccine carriers, IAVs have been shown to elicit both a cellular and humoral response against various pathogens through the display of antibody epitopes on their coat proteins. Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties.", "Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties. In the past five years, however, new, more potent broadly neutralizing antibodies that do not exhibit autoreactivity properties have been isolated from HIV-1 infected individuals, suggesting that vaccination strategies aimed at producing such broadly neutralizing antibodies may confer protection against infection. The utilization of these new, broadly neutralizing antibodies in combination with the architectural traits of IAVs have driven the current developments in the design of an inovirus-based vaccine against HIV-1. This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1. Text: Filamentous bacterial viruses are a group of thread-like viruses containing single-stranded DNA genomes.", "This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1. Text: Filamentous bacterial viruses are a group of thread-like viruses containing single-stranded DNA genomes. Collectively, they constitute the genus Inovirus in the family Inoviridae, the terms deriving from the Greek word Ίνα for filament , and they are commonly called filamentous bacteriophages. There are over 50 different known individual species of filamentous viruses; the majority of them capable of infecting Gram-negative bacteria. The complex interaction between filamentous phages and their bacterial hosts is specified by receptor organelles that are usually encoded by transmissible plasmids . One of the most intriguing features of inoviruses is that they are assembled at the host membrane, where the major capsid protein subunits replace the single-stranded DNA binding protein, and progeny virions are continuously extruded into the medium without killing the infected cell, giving rise to titers of up to 10 13 virions per milliliter of liquid culture .", "The complex interaction between filamentous phages and their bacterial hosts is specified by receptor organelles that are usually encoded by transmissible plasmids . One of the most intriguing features of inoviruses is that they are assembled at the host membrane, where the major capsid protein subunits replace the single-stranded DNA binding protein, and progeny virions are continuously extruded into the medium without killing the infected cell, giving rise to titers of up to 10 13 virions per milliliter of liquid culture . The high virus production is associated only with a mild retardation of the host's growth, which gives rise to the formation of opaque plaques on bacterial lawns. In this sense, filamentous viruses bear a resemblance to symbiotic non-pathogenic animal viruses rather than phages, the term coming from the Greek word φάγος for destroyer. Inovirus virions are flexible and slender cylindrical filaments less than 10 nm in diameter and in the order of 1000 nm in length see details in Figure 1 . Each virion has several thousand identical major capsid or coat protein subunits packaging a circular single-stranded DNA molecule.", "Inovirus virions are flexible and slender cylindrical filaments less than 10 nm in diameter and in the order of 1000 nm in length see details in Figure 1 . Each virion has several thousand identical major capsid or coat protein subunits packaging a circular single-stranded DNA molecule. Each virion also has a few specific minor proteins at each end, those at one end proximal end for attachment during infection, and those at the other end distal end for nucleation and initiation of the assembly process at the host's membrane. The number of species of inovirus isolated and characterized in different parts of the world is rather large . Among E. coli inoviruses, the best-studied and most-exploited is Ff, a group of closely related viruses fd, f1 and M13 for review see that infect male F + strains of E. coli. All Ff have almost identical DNA and protein sequences, gene organization, and most other structural parameters .", "Among E. coli inoviruses, the best-studied and most-exploited is Ff, a group of closely related viruses fd, f1 and M13 for review see that infect male F + strains of E. coli. All Ff have almost identical DNA and protein sequences, gene organization, and most other structural parameters . Fd, M13 and f1 differ in their genomes at only about 100 positions out of 6408 nucleotides for f1 and fd or 6407 nucleotides for M13. The genetics and life cycle of three viruses f1, fd and M13 have been studied extensively and we know a great deal about them. The Ff genome contains ten tightly arranged genes and a non-coding intergenic region, which contains the packaging signal , the + and − origins of DNA replication, and the major rho-dependent transcriptional terminator for recent review see . Five of the ten viral genes encode proteins found in the virion g3, g6, g8, g7 and g9 .", "The Ff genome contains ten tightly arranged genes and a non-coding intergenic region, which contains the packaging signal , the + and − origins of DNA replication, and the major rho-dependent transcriptional terminator for recent review see . Five of the ten viral genes encode proteins found in the virion g3, g6, g8, g7 and g9 . Genes 3 and 6 are found on the proximal end of the virion and are essential for infectivity and stabilization, whereas, gene 7 and gene 9 proteins, gp7 and gp9, respectively, are located at the distal end of the virion and are responsible for the initiation assembly . In the end-to-end model illustrated in Figure 1 , both the proximal gp7 and gp9 and distal gp3 and gp6 minor coat protein subunits maintain the fivefold axial symmetry of the major coat protein gp8 subunits along the virion. The life cycle of Ff filamentous viruses starts with the adsorption of the virus to the tip of the F + specific pilus of E. coli. Attachment takes place by means of an adsorption structure composed of five copies of gp3, located at the proximal end of the virion see Figure 1 , mainly through sequential binding of the gp3 N2 domain with the tip of the F pilus and the N1 domain with the periplasmic domain III of TolA for recent review see reference 4 .", "The life cycle of Ff filamentous viruses starts with the adsorption of the virus to the tip of the F + specific pilus of E. coli. Attachment takes place by means of an adsorption structure composed of five copies of gp3, located at the proximal end of the virion see Figure 1 , mainly through sequential binding of the gp3 N2 domain with the tip of the F pilus and the N1 domain with the periplasmic domain III of TolA for recent review see reference 4 . After adsorption, the virus is drawn to the surface of the cell where the major coat protein gp8 subunits become associated with the inner membrane of the cell and the circular single-stranded DNA cssDNA is released into the cytoplasm. Inside the cytoplasm, the cssDNA is converted into a parental double-stranded replicative form RF . Ff inoviruses replicate their genome by a rolling-circle mechanism. Consecutive transcription from the RF DNA, and translation as well an asymmetric single strand DNA synthesis lead to an intracellular pool of viral precursor complexes, which contain viral single-stranded DNA molecules bound with gp5 protein subunits, except a small hairpin loop that serves as the packaging signal for virion assembly .", "Ff inoviruses replicate their genome by a rolling-circle mechanism. Consecutive transcription from the RF DNA, and translation as well an asymmetric single strand DNA synthesis lead to an intracellular pool of viral precursor complexes, which contain viral single-stranded DNA molecules bound with gp5 protein subunits, except a small hairpin loop that serves as the packaging signal for virion assembly . The major coat protein gp8 subunits are synthesized with a signal peptide sequence that facilitates their transport to and insertion into the bacterial membrane, where they are cleaved by signal peptidase. After cleavage, the mature coat protein is left spanning the membrane with its C terminus in the cytoplasm and the N terminus outside the cytoplasm in the periplasm . The assembly of filamentous viruses takes place at the membrane where the gp5 subunits are replaced by gp8 subunits. The first step of the assembly sequence is the binding of the packaging signal, a site of about 30 nucleotides that forms a hairpin loop, with presumably five subunits of each of the minor coat proteins gp7 and gp9 .", "The assembly of filamentous viruses takes place at the membrane where the gp5 subunits are replaced by gp8 subunits. The first step of the assembly sequence is the binding of the packaging signal, a site of about 30 nucleotides that forms a hairpin loop, with presumably five subunits of each of the minor coat proteins gp7 and gp9 . Virus assembly proceeds as single-stranded DNA passes through the membrane and more mature coat protein gp8 subunits are added until the entire DNA molecule is packaged. At the distal end of the virion, five copies of each of gp6 and gp3 are added and the complete virion is released into the medium . The assembly of inoviruses on the bacterial inner membrane is a harmonized sequential process that involves a variety of interactions between viral-encoded proteins gp1, gp4 and gp11 and host-encoded proteins, without killing the host bacterium reviewed by . The structure of the Ff virus has been extensively studied by a number of laboratories in the last five decades.", "The assembly of inoviruses on the bacterial inner membrane is a harmonized sequential process that involves a variety of interactions between viral-encoded proteins gp1, gp4 and gp11 and host-encoded proteins, without killing the host bacterium reviewed by . The structure of the Ff virus has been extensively studied by a number of laboratories in the last five decades. However, despite all of the efforts, the structure has not been completely determined and some critical questions remain unanswered. The major difficulty is that these viruses cannot be crystallized. They can be oriented in fibers suitable for X-ray fiber diffraction studies, but these are not crystals. Interpretations of the fiber diffraction patterns together with a number of physicochemical measurements, have shown that the major coat protein gp8 subunits have a five-start helical symmetry 5-fold rotation axis and are referred to as Class I strictly based on the fundamental symmetry of the protein subunits helices as determined by fiber diffraction reviewed by .", "They can be oriented in fibers suitable for X-ray fiber diffraction studies, but these are not crystals. Interpretations of the fiber diffraction patterns together with a number of physicochemical measurements, have shown that the major coat protein gp8 subunits have a five-start helical symmetry 5-fold rotation axis and are referred to as Class I strictly based on the fundamental symmetry of the protein subunits helices as determined by fiber diffraction reviewed by . On the other hand, the structure of the packaged ssDNA molecule in the virion, including its helical symmetry and the interactions with the protein sheath, is one of the least understood aspects. The structure of the DNA inside these viruses cannot be determined by conventional X-ray fiber diffraction techniques, partly because of the low DNA content in the virions. Theoretical studies have demonstrated that the single-stranded DNA molecule is uniquely packaged inside the Ff virion by predominant electrostatic interactions . A 3D scale schematic model of an end-to-end Ff fd inoviral virion.", "Theoretical studies have demonstrated that the single-stranded DNA molecule is uniquely packaged inside the Ff virion by predominant electrostatic interactions . A 3D scale schematic model of an end-to-end Ff fd inoviral virion. The model is based on published physical data including the determined helical parameters of the major coat protein gp8 and the X-ray structure of the N1-N2 domains of the minor coat protein gp3. The model shows the relative location of the circular single-stranded DNA cssDNA genome 6408 nucleotides long, illustrated as blue ribbons , some structural details of the outer virion capsid major coat protein pg8 and the four minor coat proteins gp6, gp3, gp7 and gp9 present at the ends of the virion. On top, a digital scanning transmission electron micrograph STEM of unstained fd virus, prepared by the wet-film technique according to previously established procedures of the Brookhaven STEM facility . The ends of one complete virion are designated by arrows.", "On top, a digital scanning transmission electron micrograph STEM of unstained fd virus, prepared by the wet-film technique according to previously established procedures of the Brookhaven STEM facility . The ends of one complete virion are designated by arrows. The data were collected in collaboration with L.A. Day and J. S. Wall at the Brookhaven National Laboratory, Upton New York. Under these STEM conditions fd virions are about 8800 Å long and about 65 Å in diameter . In the middle, a proposed end-to-end scale 3D diagram of fd virion is presented. The entire fd virion is composed of about 2700 subunits of gp8 with the exception of its two ends.", "In the middle, a proposed end-to-end scale 3D diagram of fd virion is presented. The entire fd virion is composed of about 2700 subunits of gp8 with the exception of its two ends. Architectural details of an axial slab 176 Å long about 1/50 of the virion length consisting entirely of subunits of major coat protein gp8. The structure of the 50-amino-acid-long and extended α-helical gp8, shown below in both surface and ribbon images, is presented in the virion model as a cylindrical stack of 25 gray disks about 70 Å long and 10 Å in diameter. The images of gp8 were derived from coordinates of RCSB PDB database accession number 2cOW using PyMOL . The gp8 subunits are arranged with a helical symmetry that includes a two-fold screw axis and a five-fold rotation axis, consisting of two pentamers of pg8 35 .", "The images of gp8 were derived from coordinates of RCSB PDB database accession number 2cOW using PyMOL . The gp8 subunits are arranged with a helical symmetry that includes a two-fold screw axis and a five-fold rotation axis, consisting of two pentamers of pg8 35 . The two pentamers are architecturally related to each other by a translation of about 16 Å along the virion axis and a rotation of 36° about the axis . The proximal end of the virion, shown on the left, is composed of five copies of each of the minor coat proteins gp6 and gp3 for a recent review see . The proximal end is modeled based on partial information known about the structures of gp6 and gp3. Specifically, the N-terminal portion of gp6 was modeled following the helical parameters of gp8, based on protein sequence homology between the two .", "The proximal end is modeled based on partial information known about the structures of gp6 and gp3. Specifically, the N-terminal portion of gp6 was modeled following the helical parameters of gp8, based on protein sequence homology between the two . Five copies of gp3 subunits were modeled based on structural information of the N1-N2 domains. The images of the N1-N2 domains of gp3 are shown below and were derived from coordinates of RCSB PDB database accession number 1g3p using PyMOL. The domain organization of gp3 is also shown. The distal end of the virion right consists of five copies of each minor coat proteins gp7 and gp9, modeled following the helical parameters of gp8 according to a previously published model .", "The domain organization of gp3 is also shown. The distal end of the virion right consists of five copies of each minor coat proteins gp7 and gp9, modeled following the helical parameters of gp8 according to a previously published model . The easy genetic manipulation of inoviruses and the possibility of inserting random oligonucleotides into their genome set the foundation for inovirus display phage display technology . Expression of these genetically modified inoviruses results in the presentation of oligopeptides as fusion proteins on the surface of the virion and are herein termed IAVs for inovirus-associated vectors. IAVs can be modified to express an oligopeptide on either all or on some copies of a particular capsid protein. One possibility is to insert an oligonucleotide sequence of interest in the viral genome to create a fusion with capsid protein gp3, gp7, gp8 or gp9, so that the oligopeptide is displayed on every copy of the capsid protein.", "IAVs can be modified to express an oligopeptide on either all or on some copies of a particular capsid protein. One possibility is to insert an oligonucleotide sequence of interest in the viral genome to create a fusion with capsid protein gp3, gp7, gp8 or gp9, so that the oligopeptide is displayed on every copy of the capsid protein. Alternatively, a phagemid vector can be used, which carries an extra copy of a capsid protein to which the oligonucleotide is fused. Coinfection of bacterial hosts with the phagemid vector and a replication deficient helper phage, that carries the wild type capsid protein, would result in mosaic inovirus particles. That is, they will contain copies of both the wild type coat protein and the recombinant protein that contains the oligopeptide of interest . Non-mosaic and mosaic IAVs that display a peptide on gp3 or gp8 have been recently reviewed while IAVs that display a peptide on gp7 or gp9 have been reviewed elsewhere .", "That is, they will contain copies of both the wild type coat protein and the recombinant protein that contains the oligopeptide of interest . Non-mosaic and mosaic IAVs that display a peptide on gp3 or gp8 have been recently reviewed while IAVs that display a peptide on gp7 or gp9 have been reviewed elsewhere . In contrast to the other four-capsid proteins, gp6 capsid protein has only been utilized for the production of mosaic virions . Figure 2 illustrates the display of an antigen on each of the five capsid proteins of an IAV as indicated in published literature. It also introduces a new terminology to denote the gene to which the oligopeptides are fused to and whether the virion is a mosaic. To display many copies of an oligopeptide on an IAV, the ideal capsid protein to utilize is gp8.", "It also introduces a new terminology to denote the gene to which the oligopeptides are fused to and whether the virion is a mosaic. To display many copies of an oligopeptide on an IAV, the ideal capsid protein to utilize is gp8. The resulting non-mosaic IAV can display a peptide on each of the approximately 2700 copies of gp8 on its capsid surface. The tradeoff, however, is a significant limitation in the size of the peptide: only peptides up to 6 amino acids may be displayed without distorting the assembly of the virus see Figures 2 and 3 . This size restriction of the displayed peptide may be overcome by generating a mosaic IAV that displays the foreign peptide in only a minority of gp8 on the viral surface . With regards to the display of peptides on gp3, it is possible through mosaic IAVs to present even a whole protein on the viral surface .", "This size restriction of the displayed peptide may be overcome by generating a mosaic IAV that displays the foreign peptide in only a minority of gp8 on the viral surface . With regards to the display of peptides on gp3, it is possible through mosaic IAVs to present even a whole protein on the viral surface . Although in such a case the protein is expected to be present in up to five copies per virion, studies show that virions carry none, or just one copy of the protein on their surface . Random Peptide Libraries RPL is one common application of IAVs, where random oligopeptides are displayed on different clones of an inovirus particle. The vast diversity of an RPL depends on the size of the oligopeptide where the complexity of an RPL increases exponentially as the size of the oligopeptide increases. RPLs can subsequently be used in many applications including the identification of peptide ligands by receptors, the mapping of substrate sites for enzymes, and the creation of antibody peptide libraries.", "The vast diversity of an RPL depends on the size of the oligopeptide where the complexity of an RPL increases exponentially as the size of the oligopeptide increases. RPLs can subsequently be used in many applications including the identification of peptide ligands by receptors, the mapping of substrate sites for enzymes, and the creation of antibody peptide libraries. These applications are reviewed elsewhere . Inovirus display technology has also been used for epitope mapping and vaccine design purposes. RPLs can be used to characterize the epitope of an antibody of interest through biopanning with a monoclonal antibody of interest, which can result in the isolation of mimotopes; these are oligopeptides that mimic the native antibody epitope. The selected recombinant inoviruses that carry mimotopes can then be isolated in order to determine the DNA and amino acid sequence of the displayed oligopeptides.", "RPLs can be used to characterize the epitope of an antibody of interest through biopanning with a monoclonal antibody of interest, which can result in the isolation of mimotopes; these are oligopeptides that mimic the native antibody epitope. The selected recombinant inoviruses that carry mimotopes can then be isolated in order to determine the DNA and amino acid sequence of the displayed oligopeptides. DNA sequencing of such inserts as well as structure prediction analysis can potentially identify the previously unknown target of an antibody. Besides antibody characterization, inoculation of recombinant inoviruses isolated through this approach can potentially be used as vaccine carriers. For example, if a neutralizing antibody against a pathogen is used to screen an RPL, the selected peptides fused to inoviruses would mimic the original antibody epitope. Vaccination of animals with these inoviruses could ultimately induce the production of similar antibodies by the vaccinated individual, offering protection from infection against the pathogen.", "For example, if a neutralizing antibody against a pathogen is used to screen an RPL, the selected peptides fused to inoviruses would mimic the original antibody epitope. Vaccination of animals with these inoviruses could ultimately induce the production of similar antibodies by the vaccinated individual, offering protection from infection against the pathogen. Inovirus display technology has been successfully applied in the development of vaccines against various pathogens Tables 1 and 2 . The potential for inovirus display technology to facilitate the mapping of antibody epitopes is of great importance, especially in the case of HIV-1. Epitopes of broadly neutralizing monoclonal anti-HIV-1 antibodies could be rare, vulnerable spots on the surface of a frequently mutating virus such as HIV-1 and therefore, their identification and further study could lead to new drug therapies or vaccine targets. This review focuses particularly on the applications of inovirus display technology that utilizes capsid proteins gp3 and gp8, as those have been used in vaccine development.", "Epitopes of broadly neutralizing monoclonal anti-HIV-1 antibodies could be rare, vulnerable spots on the surface of a frequently mutating virus such as HIV-1 and therefore, their identification and further study could lead to new drug therapies or vaccine targets. This review focuses particularly on the applications of inovirus display technology that utilizes capsid proteins gp3 and gp8, as those have been used in vaccine development. Schematic representations of antigen display on the surface of Ff inovirus-associated vectors IAVs . Foreign antigens are shown as red spheres. The designation on the left denotes the inoviral gene, which can be genetically modified to express an antigen on the outer architecture of the virion. IAVs that contain both the wild type and antigen display capsid proteins are designated by \"m\" which indicates that the virion is a mosaic.", "The designation on the left denotes the inoviral gene, which can be genetically modified to express an antigen on the outer architecture of the virion. IAVs that contain both the wild type and antigen display capsid proteins are designated by \"m\" which indicates that the virion is a mosaic. Each Ff virion contains about 2700 copies of major capsid protein gp8, and five copies of each of the minor capsid proteins, gp3, gp6, gp7 and gp9. inovirus-associated vector bottom showing the major coat protein gp8 subunits arranged with a combined five-fold rotation axis and an approximate two-fold screw axis . Right, the corresponding surface lattices, identical to those previously published . The lattice diagrams show the relative position of each gp8 subunit on the outer virion surface.", "Right, the corresponding surface lattices, identical to those previously published . The lattice diagrams show the relative position of each gp8 subunit on the outer virion surface. The five gp8 subunits of each of the two interlocking pentamers constituting the helical symmetry of the virion are indicated by blue and green dots respectively. The relative virion surface area about 1400 Å 2 associated with each gp8 subunit is marked in yellow. The virion perimetrical azimuthial distance is calculated based on a virion diameter of about 65 Å. The displayed antigens, represented by red spheres, are arranged on the surface of the Ff.g8 inovirus-associated vector according to helical symmetry of the virion outer architecture bottom .", "The virion perimetrical azimuthial distance is calculated based on a virion diameter of about 65 Å. The displayed antigens, represented by red spheres, are arranged on the surface of the Ff.g8 inovirus-associated vector according to helical symmetry of the virion outer architecture bottom . IAVs are effective vaccine carriers and, as shown in Tables 1 and 2 , they have been used successfully in numerous vaccine development studies. They have been utilized in the development of vaccines against a wide variety of viral, protozoan and worm parasites and also against non-infectious diseases like Alzheimer and various types of cancer. All these attempts can be divided in two main sub-categories. The first sub-category, inovirus display technology was used to screen RPLs with monoclonal antibodies and then to select the immunogenic peptides of interest.", "All these attempts can be divided in two main sub-categories. The first sub-category, inovirus display technology was used to screen RPLs with monoclonal antibodies and then to select the immunogenic peptides of interest. The selected peptide was used as a vaccine in its soluble form or in conjugation with carrier proteins 55,66,70,81-84, 86-88,90-92,98 . In the second sub-category, similar to the first sub-category, inovirus display technology has been used for epitope mapping and isolation, but in this case, inoviruses were also used as the vaccine carriers for the immunogenic peptides 56, 57, . In contrast to IAVs, soluble peptides have the disadvantage of being less stable than the same peptides fused to inoviral particles. Soluble peptides have a flexible 3D structure and thus, do not always retain the 3D structure of the desirable epitope.", "In contrast to IAVs, soluble peptides have the disadvantage of being less stable than the same peptides fused to inoviral particles. Soluble peptides have a flexible 3D structure and thus, do not always retain the 3D structure of the desirable epitope. As a result, soluble peptides, unlike inovirus-bound peptides, are less capable of inducing the production of the desirable antibodies . Additionally, the inoviral vectors displaying peptides are highly immunogenic. Their high immunogenicity is reinforced by the ability of inoviruses to display multiple copies of peptides on their surface. Additionally, inoviruses are known for their structural simplicity, which allows the immune system to focus selectively on the displayed peptides and not on the viral carrier .", "Their high immunogenicity is reinforced by the ability of inoviruses to display multiple copies of peptides on their surface. Additionally, inoviruses are known for their structural simplicity, which allows the immune system to focus selectively on the displayed peptides and not on the viral carrier . Furthermore, since inoviruses can replicate in E. coli cultures, the cost of vaccine production is low. In summary, IAVs can be used as efficient and cost effective vaccine carriers. A large number of research studies have focused on the application of inovirus-based vaccines against infectious diseases. A common approach in many of these efforts was to vaccinate animals with inoviruses and to then challenge them with specific pathogens, in order to assess the level of protection against the pathogens.", "A large number of research studies have focused on the application of inovirus-based vaccines against infectious diseases. A common approach in many of these efforts was to vaccinate animals with inoviruses and to then challenge them with specific pathogens, in order to assess the level of protection against the pathogens. Three of these studies focused on immunization against viral parasites. In 1997, Bastien et al. fused a 15-mer linear epitope of Human Respiratory Syncytial Virus RSV glycoprotein G on inovirus gp3 and used the recombinant inovirus to vaccinate mice . This resulted in a specific humoral response, with the vaccinated animals having complete protection from challenge with the RSV virus . In 2000, Grabowska et al.", "This resulted in a specific humoral response, with the vaccinated animals having complete protection from challenge with the RSV virus . In 2000, Grabowska et al. used monoclonal antibodies against Herpes Simplex Virus type 2 HSV-2 glycoprotein G2 to screen 15-mer RPLs . The selected recombinant inoviruses were used to vaccinate mice, resulting in a specific humoral response. A high survival rate of vaccinated mice after challenge with a lethal dose of the virus was observed, and the level of protection was proportional to the dose of the inoviral vaccine . Additionally, in 2000, Yu et al. used monoclonal antibodies against the surface glycoprotein of Neurotropic Murine Coronavirus to screen various RPLs .", "Additionally, in 2000, Yu et al. used monoclonal antibodies against the surface glycoprotein of Neurotropic Murine Coronavirus to screen various RPLs . A selected clone displaying a 13-mer peptide induced a humoral immune response but no cellular response in mice. Even so, after lethal virus challenge, three out of six mice survived. . Besides viral infections, inoviral vaccine research has also been applied for systemic candidiasis, a fungal infection caused by Candida albicans. In two separate studies, a specific six amino acid peptide epitope of the fungal heat shock protein 90 was displayed as a fusion with the inoviral coat protein gp8 .", "Besides viral infections, inoviral vaccine research has also been applied for systemic candidiasis, a fungal infection caused by Candida albicans. In two separate studies, a specific six amino acid peptide epitope of the fungal heat shock protein 90 was displayed as a fusion with the inoviral coat protein gp8 . After infection with the parasite, mice immunized with the recombinant inoviruses had fewer colony forming units of Candida albicans in the kidney and a longer lifespan . The use of inovirus as a vaccine carrier was particularly successful against Taenia solium, a parasitic worm that uses pigs as intermediate hosts and causes neurocysticercosis, a parasitic disease of the central nervous system, in humans . In 2004, Manoutsarian et al. fused four antigenic peptides GK1, KETc1, KETc7, KETc12 to inoviruses and the cocktail of recombinant inoviruses was used to vaccinate pigs; as a result, a specific cellular response was induced.", "In 2004, Manoutsarian et al. fused four antigenic peptides GK1, KETc1, KETc7, KETc12 to inoviruses and the cocktail of recombinant inoviruses was used to vaccinate pigs; as a result, a specific cellular response was induced. Vaccination of pigs protected them against challenge with the pathogen: 1/3 of pigs were totally protected and 2/3 had reduced number of cysticerci . Based on these results, large-scale vaccination of 1047 rural pigs in 16 villages of central Mexico was conducted in 2008. The immunization was successful since the vaccine conferred significant protection against the parasite. Furthermore, this inovirus-based vaccine was more economical compared with to another vaccine made of synthetic peptides.", "The immunization was successful since the vaccine conferred significant protection against the parasite. Furthermore, this inovirus-based vaccine was more economical compared with to another vaccine made of synthetic peptides. The study was particularly important, not only due to the large scale vaccination attempt with inoviruses, but also due to its effectiveness in significantly reducing the number of cysticerci in the vaccinated animals . Efforts were also made to develop vaccines against the worm Schistosoma japonicum. In 2004, Tang et al. screened a 12-mer RPL with polyclonal serum from infected mice . The selected recombinant inoviruses induced a specific humoral response, which conferred partial protection from parasite challenge in the vaccinated mice .", "screened a 12-mer RPL with polyclonal serum from infected mice . The selected recombinant inoviruses induced a specific humoral response, which conferred partial protection from parasite challenge in the vaccinated mice . Following this study, in 2006, Wang et al. used the monoclonal antibody SSj14, which targets the parasite to screen a 12-mer RPL . The recombinant inoviruses induced both humoral and cellular responses that significantly protected the vaccinated mice against the worm infection . Additionally, in 2006, Wu et al. used polyclonal serum from infected rabbits to screen a 12-mer RPL . A humoral response was induced in the vaccinated mice, which conferred partial protection from the parasite .", "used polyclonal serum from infected rabbits to screen a 12-mer RPL . A humoral response was induced in the vaccinated mice, which conferred partial protection from the parasite . In 2008, Villa-Mancera et al., produced a vaccine against Fasciola hepatica, by screening a previously constructed 12-mer inovirus RLP with an anti-cathepsin L monoclonal antibody . Although immunization of sheep with the selected recombinant inoviruses induced a weak, specific humoral response after challenge with the parasite, the vaccinated animals had a remarkable reduction in worm burden compared to controls . An inovirus-based vaccine has been constructed against the parasitic worm Trichinella spiralis. In this case, Gu et al.", "An inovirus-based vaccine has been constructed against the parasitic worm Trichinella spiralis. In this case, Gu et al. used a monoclonal antibody against rTs87 antigen to screen a 12-mer RPL . As a result, a humoral response was induced and the vaccinated mice gained partial protection after challenge against the parasite . In summary, the results of the above studies show that the construction of an efficient inovirus-based vaccine that confers protection to the vaccinated animals against the infectious pathogen is achievable through the induction of humoral or cellular immune response or both. As previously mentioned, inovirus display technology has also been used for the design of vaccines against non-infectious diseases and in some cases, the capability of the vaccine to limit the progression of the disease was evaluated.", "In summary, the results of the above studies show that the construction of an efficient inovirus-based vaccine that confers protection to the vaccinated animals against the infectious pathogen is achievable through the induction of humoral or cellular immune response or both. As previously mentioned, inovirus display technology has also been used for the design of vaccines against non-infectious diseases and in some cases, the capability of the vaccine to limit the progression of the disease was evaluated. In 2005, Fang et al. displayed an epitope of the Melanoma Antigen A1 MAGE A1 in the surface of inovirus fused on gp8 . This resulted in an induction of cellular immune response against the melanoma tumor and in the significant inhibition of tumor growth in the vaccinated mice. In addition, there was an important increase in the survival rate of vaccinated animals .", "This resulted in an induction of cellular immune response against the melanoma tumor and in the significant inhibition of tumor growth in the vaccinated mice. In addition, there was an important increase in the survival rate of vaccinated animals . Similar results were obtained in 2002 by Wu et al. in an effort to design a vaccine against murine mastocytoma P815 . An epitope of the P1A tumor antigen was fused to the inoviral surface and the vaccinated mice gained significant protection against tumor growth. Moreover, there was a significant increase in survival rate due to an anti-tumor cellular response that was induced .", "An epitope of the P1A tumor antigen was fused to the inoviral surface and the vaccinated mice gained significant protection against tumor growth. Moreover, there was a significant increase in survival rate due to an anti-tumor cellular response that was induced . Some important efforts have also been made for the design of an inovirus-based vaccine against Alzheimer's disease. The main target of these vaccines was the induction of antibodies against β-amyloid plaques. In these studies, the antigenic epitope that was displayed in the inoviral surface was the epitope EGFR, which consists of the four amino acids E, G, F and R and it is part of the β-amyloid peptide. In mice immunized with recombinant inoviruses, the researchers observed a reduction in β-amyloid plaque burden and a specific humoral response .", "In these studies, the antigenic epitope that was displayed in the inoviral surface was the epitope EGFR, which consists of the four amino acids E, G, F and R and it is part of the β-amyloid peptide. In mice immunized with recombinant inoviruses, the researchers observed a reduction in β-amyloid plaque burden and a specific humoral response . Collectively, these studies show that it is possible to protect vaccinated animals against disease progression, thus alluding to the promising use of such vaccines against non-infectious diseases in humans. In summary, the utilization of inoviral vectors for vaccine development against infectious and non-infectious non-HIV-1 diseases has produced significant and promising results. First, in the majority of cases, the vaccine was successful since it induced specific humoral or cellular response, or both. Furthermore, in many cases, there was an attempt to evaluate the efficacy of the vaccine after challenge against the pathogen in vaccinated animals.", "First, in the majority of cases, the vaccine was successful since it induced specific humoral or cellular response, or both. Furthermore, in many cases, there was an attempt to evaluate the efficacy of the vaccine after challenge against the pathogen in vaccinated animals. In all cases, the vaccine could provide partial, significant or even complete protection against the pathogen. This established the effectiveness of the use of inoviruses as vaccine vectors. Our knowledge of HIV-1 neutralization epitopes initiated from the isolation of several neutralizing monoclonal antibodies 2F5, 4E10, b12 and 2G12 that were described between 1993 and 1994 . Thus far, neutralizing monoclonal antibodies have been found to target four major epitopes on the HIV-1 envelope gp41 and gp120 glycoproteins .", "Our knowledge of HIV-1 neutralization epitopes initiated from the isolation of several neutralizing monoclonal antibodies 2F5, 4E10, b12 and 2G12 that were described between 1993 and 1994 . Thus far, neutralizing monoclonal antibodies have been found to target four major epitopes on the HIV-1 envelope gp41 and gp120 glycoproteins . These monoclonal antibodies target the MPER epitope of gp41 monoclonal antibodies 2F5, 4E10, M66.6, CAP206-CH12 and 10e8 ; the V1V2-glycan of gp120 PG9, PG16, CH01-04 and PGT 141-145 ; the glycan dependent site of the gp120 V3 loop PGT121-123, PGT125-131 and PGT135-137 ; and the CD4-binding site b12, HJ16, CH103-106, VRC01-03, VRC-PG04, VRC-PG04b, VRC-CH30-34, 3BNC117, 3BNC60, NIH45-46, 12A12, 12A21, 8ANC131, 8ANC134, INC9 and IB2530 102, 114, . Monoclonal antibody 2G12 targets the surface glycans on the outer domain of gp120 that is distinct from the four major epitope target sites described above . The inovirus display technology has also been applied in vaccination strategies against HIV-1 . However, unlike the successful development of vaccines against non-HIV-1 parasites, these efforts failed.", "The inovirus display technology has also been applied in vaccination strategies against HIV-1 . However, unlike the successful development of vaccines against non-HIV-1 parasites, these efforts failed. In all published studies see Table 1 , the HIV-1 inovirus-display vaccines were made utilizing the broadly neutralizing antibodies 2F5, 2G12 and b12 . The first study for the construction of a vaccine against HIV-1 using inovirus display technology was performed in 1993 by Keller et al., using the 447-52D monoclonal antibody to screen a 15-mer RPL . Vaccination of selected recombinant inoviruses in rabbits resulted in the induction of type-specific neutralizing antibodies . A few years later, in 2001, Zwick et al.", "Vaccination of selected recombinant inoviruses in rabbits resulted in the induction of type-specific neutralizing antibodies . A few years later, in 2001, Zwick et al. used b12 monoclonal antibody to screen a variety of linear and constrained RPLs . However, the vaccination in mice and rabbits with the selected recombinant inoviruses did not result in the production of b12-like antibodies at detectable levels . The same antibody was used in 2005 by Dorgham et al. , in order to screen a 15-mer RPL, and the selected mimotopes were fused to the capsid of inoviruses which were then used to vaccinate mice . The induced antibodies could bind gp160 but they did not have neutralizing potency .", ", in order to screen a 15-mer RPL, and the selected mimotopes were fused to the capsid of inoviruses which were then used to vaccinate mice . The induced antibodies could bind gp160 but they did not have neutralizing potency . In another study in 2007, Wilkinson et al. screened a 9-mer and a constrained 10-mer library with antibody 5145A . This was the only case in which the selected mimotopes were fused in to small heat shock protein HSP of the archeaon Methanococcus jannaschii as a carrier protein. Following vaccination of HSP-mimotopes in rabbits, anti-gp120 antibodies without neutralizing potency were produced . The 2G12 antibody was used for the first time in 2008 by Menendez et al.", "Following vaccination of HSP-mimotopes in rabbits, anti-gp120 antibodies without neutralizing potency were produced . The 2G12 antibody was used for the first time in 2008 by Menendez et al. for the screening of a variety of linear and constrained RPLs . Nevertheless, vaccination of selected inoviruses in rabbits induced the production of antibodies that could not bind to gp120 . More recently, in 2011, Rodriguez et al. used the 2F5 antibody to screen a 12-mer and a constrained 7-mer RPL . Vaccination in mice and rabbits led to the production of non-neutralizing antibodies . In most of these studies, the use of inoviral vectors resulted in the induction of a specific humoral response.", "Vaccination in mice and rabbits led to the production of non-neutralizing antibodies . In most of these studies, the use of inoviral vectors resulted in the induction of a specific humoral response. However, the sera of the vaccinated animals did not have broadly neutralizing ability. Besides monoclonal antibodies, polyclonal sera from HIV-1-infected patients were also used for the screening RPLs Table 1 51, 53, 63 . This approach carries a degree of uncertainty, since it is based on the premise that the polyclonal sera will contain at least one broadly neutralizing monoclonal anti-HIV-1 antibody, meaning that a new neutralizing epitope against it can be isolated from the RPL. It is suggested that long-term non-progressors HIV-1-infected patients who remain asymptomatic for a long time are more likely to produce neutralizing antibodies in comparison to AIDS patients, and it is suggested that these neutralizing antibodies in the serum of long-term non-progressors may contribute to the control of viral load .", "This approach carries a degree of uncertainty, since it is based on the premise that the polyclonal sera will contain at least one broadly neutralizing monoclonal anti-HIV-1 antibody, meaning that a new neutralizing epitope against it can be isolated from the RPL. It is suggested that long-term non-progressors HIV-1-infected patients who remain asymptomatic for a long time are more likely to produce neutralizing antibodies in comparison to AIDS patients, and it is suggested that these neutralizing antibodies in the serum of long-term non-progressors may contribute to the control of viral load . However, this hypothesis has been questioned . Polyclonal serum for the screening of RPLs was used for the first time in 1999 by Scala et al., who screened both linear and constrained 9-mer RPLs . After that, vaccination of selected inoviruses in mice led to the production of neutralizing antibodies. A few years later, in 2007, Rodriguez et al.", "After that, vaccination of selected inoviruses in mice led to the production of neutralizing antibodies. A few years later, in 2007, Rodriguez et al. used polyclonal serum to screen a 7-mer, a 12-mer and a constrained 7-mer RPL . Vaccination in mice induced the production of antibodies that could bind to gp41, but no information was provided about their neutralization potency . Additionally, in 2007, Humbert et al. used polyclonal serum to screen a 7-mer, a 12-mer and a constrained 7-mer RPL . Vaccination of selected recombinant inoviruses in mice induced the production of neutralizing antibodies .", "used polyclonal serum to screen a 7-mer, a 12-mer and a constrained 7-mer RPL . Vaccination of selected recombinant inoviruses in mice induced the production of neutralizing antibodies . The screening of the same RPLs using polyclonal serum from a monkey infected with a Simian-Human Immunodeficiency Virus SHIV was performed by the same group. In this study, vaccination in mice was performed using the prime-boost strategy: DNA vaccine as prime encoding gp160 and a cocktail of recombinant inoviruses as boost. The result was the induction of neutralizing antibodies . In 2013, Gazarian et al. screened a linear 12-mer and constrained 7-mer RPLs using sera from HIV-infected individuals .", "The result was the induction of neutralizing antibodies . In 2013, Gazarian et al. screened a linear 12-mer and constrained 7-mer RPLs using sera from HIV-infected individuals . Vaccination in rabbits resulted in the production of antibodies that bind gp160 . In 2001, non-human primates were used by Chen et al., as an animal model for vaccination with recombinant inoviruses . This group performed screening of a 9-mer and a constrained 9-mer RPL with polyclonal serum isolated from an infected donor. After that, the vaccination of selected inoviruses was performed in rhesus macaques. As a result, sera from the vaccinated macaques exhibited neutralizing activity.", "After that, the vaccination of selected inoviruses was performed in rhesus macaques. As a result, sera from the vaccinated macaques exhibited neutralizing activity. Furthermore, the vaccinated macaques were not protected from infection, but four out of five animals were able to control the viral load after challenge against the pathogenic SHIV89.6PD virus. This was a very important result, which underlines the potential of the method. Additionally, no specific CTL response was detected, implying that the control of viral load was an exclusive result of humoral response . Since the first attempt to induce the production of anti-HIV-1 neutralizing antibodies using inovirus-based vaccines, all efforts to date have not led to the production of broadly neutralizing antibodies in vaccinated animals.", "Additionally, no specific CTL response was detected, implying that the control of viral load was an exclusive result of humoral response . Since the first attempt to induce the production of anti-HIV-1 neutralizing antibodies using inovirus-based vaccines, all efforts to date have not led to the production of broadly neutralizing antibodies in vaccinated animals. This failure could be to a certain extent explained by the usage of the \"old generation\" monoclonal antibodies 2F5, 2G12 and b12 which were shown to demonstrate autoreactivity properties in vitro studies . In 2010, Verkoczy et al. demonstarted in an in vivo study that Pre-B cells expressing 2F5-like antibodies were unable to maturate in mice, suggesting a triggering of immunological tolerance due to the autoreactive properties of the 2F5-like antibody . Collectively, these studies implied that the screening RPLs using 2F5, 2G12 and b12 could result in inoviral-based vaccines that could trigger immunological tolerance in vaccinated animals.", "demonstarted in an in vivo study that Pre-B cells expressing 2F5-like antibodies were unable to maturate in mice, suggesting a triggering of immunological tolerance due to the autoreactive properties of the 2F5-like antibody . Collectively, these studies implied that the screening RPLs using 2F5, 2G12 and b12 could result in inoviral-based vaccines that could trigger immunological tolerance in vaccinated animals. It is important to note, however, that the lack of autoreactivity properties for several of the \"next generation\" broadly neutralizing antibodies 10e8, PG9, PG16, VRC01-03, VRC-PG04, VRC-PG04b, and VRC-CH30-34 could solve the autoreactivity problems encountered by 2F5, 2G12, and b12. Despite the fact that the recent isolation of new broadly neutralizing antibodies against HIV-1 has focused the attention of HIV-1 vaccine development on the induction of a humoral anti-HIV-1 response, recent results underline the importance of a cellular anti-HIV-1 response as well . The experimental results concerning inovirus-based vaccines in non-HIV-1 diseases prove that inoviruses can also induce a strong specific cellular response Tables 1 and 2 ; this property makes them great candidates as vectors for HIV-1 vaccine design. The ability of inoviruses to induce a cellular immune response was first demonstrated in 2000 by DeBerardinis et al.", "The experimental results concerning inovirus-based vaccines in non-HIV-1 diseases prove that inoviruses can also induce a strong specific cellular response Tables 1 and 2 ; this property makes them great candidates as vectors for HIV-1 vaccine design. The ability of inoviruses to induce a cellular immune response was first demonstrated in 2000 by DeBerardinis et al. . In this work, recombinant inoviruses carrying the RT2 epitope and the pep23 epitope of HIV-1 reverse transcriptase in gp8 could induce specific cellular responses in human cell lines in vitro and in mice in vivo against the RT2 peptide. It is interesting that without the pep23 epitope, the cellular response was undetectable. This indicates that the pep23 is a CTL epitope that is necessary for cellular response, possibly because it enables internalization of the recombinant inovirus into the APCs .", "It is interesting that without the pep23 epitope, the cellular response was undetectable. This indicates that the pep23 is a CTL epitope that is necessary for cellular response, possibly because it enables internalization of the recombinant inovirus into the APCs . Therefore, a question arises of whether an epitope fused to the surface of the inovirus can induce a cellular or a humoral response or both. It is suggested that the type of immune response caused by an epitope fused on the surface of the inovirus is dependent on the length and sequence of the peptide . In 2003, Gaubin et al. demonstrated that FITC-labeled fd virions can be internalized in human EBV-B cell lines and the fd virions are successively degraded and targeted both to MHC class I and class II antigen-processing pathways .", "In 2003, Gaubin et al. demonstrated that FITC-labeled fd virions can be internalized in human EBV-B cell lines and the fd virions are successively degraded and targeted both to MHC class I and class II antigen-processing pathways . This was confirmed after endocellular localization of the labeled virions with confocal microscopy. This experiment showed that the inoviruses could be internalized in APCs even without carrying a CTL epitope, but in very low rate. For in vivo experiments however, it is possible that the requirement of a CTL epitope is critical for the induction of a cellular response . In 2011, Sartorius et al.", "For in vivo experiments however, it is possible that the requirement of a CTL epitope is critical for the induction of a cellular response . In 2011, Sartorius et al. showed that a hybrid fd virion with the anti-DEC-205 scFv antibody fragment fused on gp3 and the OVA257-264 antigenic epitope fused on gp8 can be internalized in human dendritic cells through a specific interaction between the anti-DEC205 scFv and the DEC-205 receptor . In addition, inoculation of mice with the hybrid virions induced a specific cellular response against the OVA257-264 epitope . This important characteristic of inoviral vectors to induce a cellular response was reported in only two studies aimed at developing a HIV-1 inoviral vaccine, possibly due to the complexity of detecting a cellular response and also because it is a labor intensive and time-consuming process. In 2001, Chen et al.", "This important characteristic of inoviral vectors to induce a cellular response was reported in only two studies aimed at developing a HIV-1 inoviral vaccine, possibly due to the complexity of detecting a cellular response and also because it is a labor intensive and time-consuming process. In 2001, Chen et al. attempted to detect a cellular response, but such a response was not induced in that experiment . A more recent research study related to HIV-1, which clearly demonstrates the ability of inoviruses to induce strong cellular response, was performed in 2009 by Pedroza-Roldan et al. . In this effort, an immunodominant CTL epitope of the V3 loop of gp120 residues 311-320 was expressed as fusion to gp8 of an M13 inovirus.", ". In this effort, an immunodominant CTL epitope of the V3 loop of gp120 residues 311-320 was expressed as fusion to gp8 of an M13 inovirus. A random peptide library was created by inserting mutations in certain positions of this epitope. A cocktail of inoviruses carrying the V3 loop epitope or variations of this epitope were used for the vaccination of mice and, as a result, a CTL response was induced. The most important result of this study was that the immunization induced long-lasting memory T-cell responses, which were detected seven months after a single immunization . The same vaccination also induced a strong humoral response, since the sera of vaccinated mice could neutralize five out of ten pseudoviruses from a panel .", "The most important result of this study was that the immunization induced long-lasting memory T-cell responses, which were detected seven months after a single immunization . The same vaccination also induced a strong humoral response, since the sera of vaccinated mice could neutralize five out of ten pseudoviruses from a panel . All the above experiments clearly show that the inoviruses are capable of inducing a specific cellular immune response: the ability of the inoviral vectors to induce both arms of adaptive immunity is unique and it could prove to be valuable in the development of a successful HIV-1 vaccine. In the general field of HIV-1 vaccine design, all studies for the production of anti-HIV-1 broadly neutralizing antibodies through vaccination with either soluble peptides or viral vectors have been unsuccessful. For this reason, some efforts have been directed to the induction of cellular immune response . In recent years, in HIV-1 vaccine phase I and phase II clinical trials, adenoviral vectors have been used in order induce a cellular immune response in HIV-1 vaccinated individuals .", "For this reason, some efforts have been directed to the induction of cellular immune response . In recent years, in HIV-1 vaccine phase I and phase II clinical trials, adenoviral vectors have been used in order induce a cellular immune response in HIV-1 vaccinated individuals . However, there are concerns about the safety of these viruses. In 2007, the large-scale phase IIB Merck trial was abruptly terminated because there was evidence that the individuals vaccinated with adenovirus rAd5 vector expressing gag, pol and nef became more vulnerable to HIV-1 infection in comparison to controls. It was suggested that the group with the increased risk of being infected with HIV-1 consisted of individuals who were Ad5 seropositive . Other eukaryotic viruses that infect other species and do not replicate in human cells were also tested as candidates HIV vaccine carriers and in theory are safer.", "It was suggested that the group with the increased risk of being infected with HIV-1 consisted of individuals who were Ad5 seropositive . Other eukaryotic viruses that infect other species and do not replicate in human cells were also tested as candidates HIV vaccine carriers and in theory are safer. For example, the canarypox vector was used in the RV144 phase III clinical trial, the only clinical trial that had positive results to date, offering partial protection 31% to vaccinated individuals in comparison with control . Apart from safety reasons, the use of adenovirus-based vectors has not been protective. The recent HVTV 505 phase IIB trial that used adenovirus rAd5 as a boost and DNA as prime for vaccination of 2504 human volunteers was abandoned as futile . Recently, a rhesus macaque cytomegalovirus RhCMV vector successfully induced a persisting CTL response in rhesus macaques that strongly protected the vaccinated animals from challenge against the pathogenic SIVmac239 strain.", "The recent HVTV 505 phase IIB trial that used adenovirus rAd5 as a boost and DNA as prime for vaccination of 2504 human volunteers was abandoned as futile . Recently, a rhesus macaque cytomegalovirus RhCMV vector successfully induced a persisting CTL response in rhesus macaques that strongly protected the vaccinated animals from challenge against the pathogenic SIVmac239 strain. Importantly, this study, 50% of the vaccinated animals reduced the viral load to undetectable levels . However, the design of a human version of this CMV vector could impose safety risks, since the human CMV is a persistent and pathogenic human virus . Therefore, various types of eukaryotic viral vectors are currently under investigation. These are reviewed elsewhere .", "Therefore, various types of eukaryotic viral vectors are currently under investigation. These are reviewed elsewhere . However, the use of a eukaryotic virus is accompanied by serious safety concerns. As an alternative, the use of prokaryotic viruses such as inoviruses, may be utilized which have a decisively lower safety risk to humans. Even if inoviruses could infect a eukaryotic cell, the assembly of the new prokaryotic virions cannot take place without the specific conditions that exist in the inter-membrane area of the E. coli and without the presence of the specific E. coli enzyme leader peptidase that does not exist in human cells . Furthermore, there was a phase I case study in 2006 where fd inoviruses were intravenously infused in humans for purposes unrelated to vaccination , causing no side effects or even allergic reactions in any of the eight volunteers.", "Even if inoviruses could infect a eukaryotic cell, the assembly of the new prokaryotic virions cannot take place without the specific conditions that exist in the inter-membrane area of the E. coli and without the presence of the specific E. coli enzyme leader peptidase that does not exist in human cells . Furthermore, there was a phase I case study in 2006 where fd inoviruses were intravenously infused in humans for purposes unrelated to vaccination , causing no side effects or even allergic reactions in any of the eight volunteers. To our knowledge, this is the only case where inoviruses were infused in humans . Therefore, in contrast to other viral vectors, the use of inoviruses does not impose a major safety risk to humans. This characteristic of inoviral vectors along with their capability to trigger both cellular and humoral immune responses makes them an attractive option as vaccine vectors. During the last two decades, inoviral vectors have been used in the development of vaccines against various infectious parasites and against non-infectious diseases like cancer and Alzheimer's with promising results.", "This characteristic of inoviral vectors along with their capability to trigger both cellular and humoral immune responses makes them an attractive option as vaccine vectors. During the last two decades, inoviral vectors have been used in the development of vaccines against various infectious parasites and against non-infectious diseases like cancer and Alzheimer's with promising results. While the applications of inovirus display technology in vaccine design against non-HIV-1 diseases have been mostly successful, the design of a HIV-1 vaccine development has so far been disappointing. A major obstacle has been the use of neutralizing monoclonal antibodies plagued with autoreactivity properties. Screening of RPLs with these antibodies resulted in the isolation of peptides that, as vaccine antigens, were unsuccessful in inducing a specific humoral response that would produce neutralizing antibodies. The recent isolation of antibodies such as VRC01 and 10E8 with more neutralizing breadth and potency without autoreactivity properties than the previously utilized antibodies may overcome this obstacle .", "Screening of RPLs with these antibodies resulted in the isolation of peptides that, as vaccine antigens, were unsuccessful in inducing a specific humoral response that would produce neutralizing antibodies. The recent isolation of antibodies such as VRC01 and 10E8 with more neutralizing breadth and potency without autoreactivity properties than the previously utilized antibodies may overcome this obstacle . Particularly, the induction of VRC01-like and 10E8-like antibodies could be a feasible target, since these antibodies also seem to be produced from a significant percentage of the HIV-1-infected population . While humoral responses have been well documented, cellular responses have not been assessed in most studies for the design of a HIV-1 vaccine using inoviruses. The only study in which a cellular anti-HIV-1 response was detected also reported a successful induction of a long-lasting memory CTL response seven months after a single vaccination in mice with inoviral particles , demonstrating that the induction of cellular immunity against HIV-1 using inoviruses is feasible. IAVs are advantageous in that they can induce both arms of adaptive immunity.", "The only study in which a cellular anti-HIV-1 response was detected also reported a successful induction of a long-lasting memory CTL response seven months after a single vaccination in mice with inoviral particles , demonstrating that the induction of cellular immunity against HIV-1 using inoviruses is feasible. IAVs are advantageous in that they can induce both arms of adaptive immunity. This finding could therefore be of importance in future efforts for the design of a HIV-1 vaccine. Moreover, IAVs have unique characteristics compared to other viral vectors: they are stable, they can display a peptide in multiple from few to thousands copies on their surface and such constructs are very immunogenic without the use of an adjuvant. In addition, IAVs allow the immune system to focus on a specific epitope of interest instead of the whole protein, which is of great importance, since an important aspect for successful HIV-1 vaccine design is to focus on the induction of neutralizing antibodies against the specific neutralizing epitopes while at the same time avoiding the induction of ineffective antibodies against the numerous non-neutralizing epitopes of the HIV-1 glycoproteins, which act as decoys for the immune system. Ideally, an effective HIV-1 vaccine should be able to stimulate both humoral and cellular immune responses.", "In addition, IAVs allow the immune system to focus on a specific epitope of interest instead of the whole protein, which is of great importance, since an important aspect for successful HIV-1 vaccine design is to focus on the induction of neutralizing antibodies against the specific neutralizing epitopes while at the same time avoiding the induction of ineffective antibodies against the numerous non-neutralizing epitopes of the HIV-1 glycoproteins, which act as decoys for the immune system. Ideally, an effective HIV-1 vaccine should be able to stimulate both humoral and cellular immune responses. Recently, adenoviral vectors were tested in clinical trials as HIV-1 vaccine carriers in order to induce cellular immunity, but they were shown to impose serious health risks for humans. On the other hand, IAVs are able to induce cellular immunity and at the same time they have been demonstrated to be safe for administration in animals and humans. These characteristics of IAVs, conferred by their structural and biological properties, make them effective antigen display vectors that can induce strong and specific humoral and cellular immune responses against the displayed antigen. These properties of IAVs along with newly discovered broadly neutralizing anti-HIV-1 antibodies, pave the way for the development of an effective HIV-1 vaccine." ]

[ "Inovirus-associated vectors IAVs are engineered, non-lytic, filamentous bacteriophages that are assembled primarily from thousands of copies of the major coat protein gp8 and just five copies of each of the four minor coat proteins gp3, gp6, gp7 and gp9. Inovirus display studies have shown that the architecture of inoviruses makes all coat proteins of the inoviral particle accessible to the outside. This particular feature of IAVs allows foreign antigenic peptides to be displayed on the outer surface of the virion fused to its coat proteins and for more than two decades has been exploited in many applications including antibody or peptide display libraries, drug design, and vaccine development against infectious and non-infectious diseases. As vaccine carriers, IAVs have been shown to elicit both a cellular and humoral response against various pathogens through the display of antibody epitopes on their coat proteins. Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties.", "Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties. In the past five years, however, new, more potent broadly neutralizing antibodies that do not exhibit autoreactivity properties have been isolated from HIV-1 infected individuals, suggesting that vaccination strategies aimed at producing such broadly neutralizing antibodies may confer protection against infection. The utilization of these new, broadly neutralizing antibodies in combination with the architectural traits of IAVs have driven the current developments in the design of an inovirus-based vaccine against HIV-1. This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1. Text: Filamentous bacterial viruses are a group of thread-like viruses containing single-stranded DNA genomes.", "This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1. Text: Filamentous bacterial viruses are a group of thread-like viruses containing single-stranded DNA genomes. Collectively, they constitute the genus Inovirus in the family Inoviridae, the terms deriving from the Greek word Ίνα for filament , and they are commonly called filamentous bacteriophages. There are over 50 different known individual species of filamentous viruses; the majority of them capable of infecting Gram-negative bacteria. The complex interaction between filamentous phages and their bacterial hosts is specified by receptor organelles that are usually encoded by transmissible plasmids . One of the most intriguing features of inoviruses is that they are assembled at the host membrane, where the major capsid protein subunits replace the single-stranded DNA binding protein, and progeny virions are continuously extruded into the medium without killing the infected cell, giving rise to titers of up to 10 13 virions per milliliter of liquid culture .", "The complex interaction between filamentous phages and their bacterial hosts is specified by receptor organelles that are usually encoded by transmissible plasmids . One of the most intriguing features of inoviruses is that they are assembled at the host membrane, where the major capsid protein subunits replace the single-stranded DNA binding protein, and progeny virions are continuously extruded into the medium without killing the infected cell, giving rise to titers of up to 10 13 virions per milliliter of liquid culture . The high virus production is associated only with a mild retardation of the host's growth, which gives rise to the formation of opaque plaques on bacterial lawns. In this sense, filamentous viruses bear a resemblance to symbiotic non-pathogenic animal viruses rather than phages, the term coming from the Greek word φάγος for destroyer. Inovirus virions are flexible and slender cylindrical filaments less than 10 nm in diameter and in the order of 1000 nm in length see details in Figure 1 . Each virion has several thousand identical major capsid or coat protein subunits packaging a circular single-stranded DNA molecule.", "Inovirus virions are flexible and slender cylindrical filaments less than 10 nm in diameter and in the order of 1000 nm in length see details in Figure 1 . Each virion has several thousand identical major capsid or coat protein subunits packaging a circular single-stranded DNA molecule. Each virion also has a few specific minor proteins at each end, those at one end proximal end for attachment during infection, and those at the other end distal end for nucleation and initiation of the assembly process at the host's membrane. The number of species of inovirus isolated and characterized in different parts of the world is rather large . Among E. coli inoviruses, the best-studied and most-exploited is Ff, a group of closely related viruses fd, f1 and M13 for review see that infect male F + strains of E. coli. All Ff have almost identical DNA and protein sequences, gene organization, and most other structural parameters .", "Among E. coli inoviruses, the best-studied and most-exploited is Ff, a group of closely related viruses fd, f1 and M13 for review see that infect male F + strains of E. coli. All Ff have almost identical DNA and protein sequences, gene organization, and most other structural parameters . Fd, M13 and f1 differ in their genomes at only about 100 positions out of 6408 nucleotides for f1 and fd or 6407 nucleotides for M13. The genetics and life cycle of three viruses f1, fd and M13 have been studied extensively and we know a great deal about them. The Ff genome contains ten tightly arranged genes and a non-coding intergenic region, which contains the packaging signal , the + and − origins of DNA replication, and the major rho-dependent transcriptional terminator for recent review see . Five of the ten viral genes encode proteins found in the virion g3, g6, g8, g7 and g9 .", "The Ff genome contains ten tightly arranged genes and a non-coding intergenic region, which contains the packaging signal , the + and − origins of DNA replication, and the major rho-dependent transcriptional terminator for recent review see . Five of the ten viral genes encode proteins found in the virion g3, g6, g8, g7 and g9 . Genes 3 and 6 are found on the proximal end of the virion and are essential for infectivity and stabilization, whereas, gene 7 and gene 9 proteins, gp7 and gp9, respectively, are located at the distal end of the virion and are responsible for the initiation assembly . In the end-to-end model illustrated in Figure 1 , both the proximal gp7 and gp9 and distal gp3 and gp6 minor coat protein subunits maintain the fivefold axial symmetry of the major coat protein gp8 subunits along the virion. The life cycle of Ff filamentous viruses starts with the adsorption of the virus to the tip of the F + specific pilus of E. coli. Attachment takes place by means of an adsorption structure composed of five copies of gp3, located at the proximal end of the virion see Figure 1 , mainly through sequential binding of the gp3 N2 domain with the tip of the F pilus and the N1 domain with the periplasmic domain III of TolA for recent review see reference 4 .", "The life cycle of Ff filamentous viruses starts with the adsorption of the virus to the tip of the F + specific pilus of E. coli. Attachment takes place by means of an adsorption structure composed of five copies of gp3, located at the proximal end of the virion see Figure 1 , mainly through sequential binding of the gp3 N2 domain with the tip of the F pilus and the N1 domain with the periplasmic domain III of TolA for recent review see reference 4 . After adsorption, the virus is drawn to the surface of the cell where the major coat protein gp8 subunits become associated with the inner membrane of the cell and the circular single-stranded DNA cssDNA is released into the cytoplasm. Inside the cytoplasm, the cssDNA is converted into a parental double-stranded replicative form RF . Ff inoviruses replicate their genome by a rolling-circle mechanism. Consecutive transcription from the RF DNA, and translation as well an asymmetric single strand DNA synthesis lead to an intracellular pool of viral precursor complexes, which contain viral single-stranded DNA molecules bound with gp5 protein subunits, except a small hairpin loop that serves as the packaging signal for virion assembly .", "Ff inoviruses replicate their genome by a rolling-circle mechanism. Consecutive transcription from the RF DNA, and translation as well an asymmetric single strand DNA synthesis lead to an intracellular pool of viral precursor complexes, which contain viral single-stranded DNA molecules bound with gp5 protein subunits, except a small hairpin loop that serves as the packaging signal for virion assembly . The major coat protein gp8 subunits are synthesized with a signal peptide sequence that facilitates their transport to and insertion into the bacterial membrane, where they are cleaved by signal peptidase. After cleavage, the mature coat protein is left spanning the membrane with its C terminus in the cytoplasm and the N terminus outside the cytoplasm in the periplasm . The assembly of filamentous viruses takes place at the membrane where the gp5 subunits are replaced by gp8 subunits. The first step of the assembly sequence is the binding of the packaging signal, a site of about 30 nucleotides that forms a hairpin loop, with presumably five subunits of each of the minor coat proteins gp7 and gp9 .", "The assembly of filamentous viruses takes place at the membrane where the gp5 subunits are replaced by gp8 subunits. The first step of the assembly sequence is the binding of the packaging signal, a site of about 30 nucleotides that forms a hairpin loop, with presumably five subunits of each of the minor coat proteins gp7 and gp9 . Virus assembly proceeds as single-stranded DNA passes through the membrane and more mature coat protein gp8 subunits are added until the entire DNA molecule is packaged. At the distal end of the virion, five copies of each of gp6 and gp3 are added and the complete virion is released into the medium . The assembly of inoviruses on the bacterial inner membrane is a harmonized sequential process that involves a variety of interactions between viral-encoded proteins gp1, gp4 and gp11 and host-encoded proteins, without killing the host bacterium reviewed by . The structure of the Ff virus has been extensively studied by a number of laboratories in the last five decades.", "The assembly of inoviruses on the bacterial inner membrane is a harmonized sequential process that involves a variety of interactions between viral-encoded proteins gp1, gp4 and gp11 and host-encoded proteins, without killing the host bacterium reviewed by . The structure of the Ff virus has been extensively studied by a number of laboratories in the last five decades. However, despite all of the efforts, the structure has not been completely determined and some critical questions remain unanswered. The major difficulty is that these viruses cannot be crystallized. They can be oriented in fibers suitable for X-ray fiber diffraction studies, but these are not crystals. Interpretations of the fiber diffraction patterns together with a number of physicochemical measurements, have shown that the major coat protein gp8 subunits have a five-start helical symmetry 5-fold rotation axis and are referred to as Class I strictly based on the fundamental symmetry of the protein subunits helices as determined by fiber diffraction reviewed by .", "They can be oriented in fibers suitable for X-ray fiber diffraction studies, but these are not crystals. Interpretations of the fiber diffraction patterns together with a number of physicochemical measurements, have shown that the major coat protein gp8 subunits have a five-start helical symmetry 5-fold rotation axis and are referred to as Class I strictly based on the fundamental symmetry of the protein subunits helices as determined by fiber diffraction reviewed by . On the other hand, the structure of the packaged ssDNA molecule in the virion, including its helical symmetry and the interactions with the protein sheath, is one of the least understood aspects. The structure of the DNA inside these viruses cannot be determined by conventional X-ray fiber diffraction techniques, partly because of the low DNA content in the virions. Theoretical studies have demonstrated that the single-stranded DNA molecule is uniquely packaged inside the Ff virion by predominant electrostatic interactions . A 3D scale schematic model of an end-to-end Ff fd inoviral virion.", "Theoretical studies have demonstrated that the single-stranded DNA molecule is uniquely packaged inside the Ff virion by predominant electrostatic interactions . A 3D scale schematic model of an end-to-end Ff fd inoviral virion. The model is based on published physical data including the determined helical parameters of the major coat protein gp8 and the X-ray structure of the N1-N2 domains of the minor coat protein gp3. The model shows the relative location of the circular single-stranded DNA cssDNA genome 6408 nucleotides long, illustrated as blue ribbons , some structural details of the outer virion capsid major coat protein pg8 and the four minor coat proteins gp6, gp3, gp7 and gp9 present at the ends of the virion. On top, a digital scanning transmission electron micrograph STEM of unstained fd virus, prepared by the wet-film technique according to previously established procedures of the Brookhaven STEM facility . The ends of one complete virion are designated by arrows.", "On top, a digital scanning transmission electron micrograph STEM of unstained fd virus, prepared by the wet-film technique according to previously established procedures of the Brookhaven STEM facility . The ends of one complete virion are designated by arrows. The data were collected in collaboration with L.A. Day and J. S. Wall at the Brookhaven National Laboratory, Upton New York. Under these STEM conditions fd virions are about 8800 Å long and about 65 Å in diameter . In the middle, a proposed end-to-end scale 3D diagram of fd virion is presented. The entire fd virion is composed of about 2700 subunits of gp8 with the exception of its two ends.", "In the middle, a proposed end-to-end scale 3D diagram of fd virion is presented. The entire fd virion is composed of about 2700 subunits of gp8 with the exception of its two ends. Architectural details of an axial slab 176 Å long about 1/50 of the virion length consisting entirely of subunits of major coat protein gp8. The structure of the 50-amino-acid-long and extended α-helical gp8, shown below in both surface and ribbon images, is presented in the virion model as a cylindrical stack of 25 gray disks about 70 Å long and 10 Å in diameter. The images of gp8 were derived from coordinates of RCSB PDB database accession number 2cOW using PyMOL . The gp8 subunits are arranged with a helical symmetry that includes a two-fold screw axis and a five-fold rotation axis, consisting of two pentamers of pg8 35 .", "The images of gp8 were derived from coordinates of RCSB PDB database accession number 2cOW using PyMOL . The gp8 subunits are arranged with a helical symmetry that includes a two-fold screw axis and a five-fold rotation axis, consisting of two pentamers of pg8 35 . The two pentamers are architecturally related to each other by a translation of about 16 Å along the virion axis and a rotation of 36° about the axis . The proximal end of the virion, shown on the left, is composed of five copies of each of the minor coat proteins gp6 and gp3 for a recent review see . The proximal end is modeled based on partial information known about the structures of gp6 and gp3. Specifically, the N-terminal portion of gp6 was modeled following the helical parameters of gp8, based on protein sequence homology between the two .", "The proximal end is modeled based on partial information known about the structures of gp6 and gp3. Specifically, the N-terminal portion of gp6 was modeled following the helical parameters of gp8, based on protein sequence homology between the two . Five copies of gp3 subunits were modeled based on structural information of the N1-N2 domains. The images of the N1-N2 domains of gp3 are shown below and were derived from coordinates of RCSB PDB database accession number 1g3p using PyMOL. The domain organization of gp3 is also shown. The distal end of the virion right consists of five copies of each minor coat proteins gp7 and gp9, modeled following the helical parameters of gp8 according to a previously published model .", "The domain organization of gp3 is also shown. The distal end of the virion right consists of five copies of each minor coat proteins gp7 and gp9, modeled following the helical parameters of gp8 according to a previously published model . The easy genetic manipulation of inoviruses and the possibility of inserting random oligonucleotides into their genome set the foundation for inovirus display phage display technology . Expression of these genetically modified inoviruses results in the presentation of oligopeptides as fusion proteins on the surface of the virion and are herein termed IAVs for inovirus-associated vectors. IAVs can be modified to express an oligopeptide on either all or on some copies of a particular capsid protein. One possibility is to insert an oligonucleotide sequence of interest in the viral genome to create a fusion with capsid protein gp3, gp7, gp8 or gp9, so that the oligopeptide is displayed on every copy of the capsid protein.", "IAVs can be modified to express an oligopeptide on either all or on some copies of a particular capsid protein. One possibility is to insert an oligonucleotide sequence of interest in the viral genome to create a fusion with capsid protein gp3, gp7, gp8 or gp9, so that the oligopeptide is displayed on every copy of the capsid protein. Alternatively, a phagemid vector can be used, which carries an extra copy of a capsid protein to which the oligonucleotide is fused. Coinfection of bacterial hosts with the phagemid vector and a replication deficient helper phage, that carries the wild type capsid protein, would result in mosaic inovirus particles. That is, they will contain copies of both the wild type coat protein and the recombinant protein that contains the oligopeptide of interest . Non-mosaic and mosaic IAVs that display a peptide on gp3 or gp8 have been recently reviewed while IAVs that display a peptide on gp7 or gp9 have been reviewed elsewhere .", "That is, they will contain copies of both the wild type coat protein and the recombinant protein that contains the oligopeptide of interest . Non-mosaic and mosaic IAVs that display a peptide on gp3 or gp8 have been recently reviewed while IAVs that display a peptide on gp7 or gp9 have been reviewed elsewhere . In contrast to the other four-capsid proteins, gp6 capsid protein has only been utilized for the production of mosaic virions . Figure 2 illustrates the display of an antigen on each of the five capsid proteins of an IAV as indicated in published literature. It also introduces a new terminology to denote the gene to which the oligopeptides are fused to and whether the virion is a mosaic. To display many copies of an oligopeptide on an IAV, the ideal capsid protein to utilize is gp8.", "It also introduces a new terminology to denote the gene to which the oligopeptides are fused to and whether the virion is a mosaic. To display many copies of an oligopeptide on an IAV, the ideal capsid protein to utilize is gp8. The resulting non-mosaic IAV can display a peptide on each of the approximately 2700 copies of gp8 on its capsid surface. The tradeoff, however, is a significant limitation in the size of the peptide: only peptides up to 6 amino acids may be displayed without distorting the assembly of the virus see Figures 2 and 3 . This size restriction of the displayed peptide may be overcome by generating a mosaic IAV that displays the foreign peptide in only a minority of gp8 on the viral surface . With regards to the display of peptides on gp3, it is possible through mosaic IAVs to present even a whole protein on the viral surface .", "This size restriction of the displayed peptide may be overcome by generating a mosaic IAV that displays the foreign peptide in only a minority of gp8 on the viral surface . With regards to the display of peptides on gp3, it is possible through mosaic IAVs to present even a whole protein on the viral surface . Although in such a case the protein is expected to be present in up to five copies per virion, studies show that virions carry none, or just one copy of the protein on their surface . Random Peptide Libraries RPL is one common application of IAVs, where random oligopeptides are displayed on different clones of an inovirus particle. The vast diversity of an RPL depends on the size of the oligopeptide where the complexity of an RPL increases exponentially as the size of the oligopeptide increases. RPLs can subsequently be used in many applications including the identification of peptide ligands by receptors, the mapping of substrate sites for enzymes, and the creation of antibody peptide libraries.", "The vast diversity of an RPL depends on the size of the oligopeptide where the complexity of an RPL increases exponentially as the size of the oligopeptide increases. RPLs can subsequently be used in many applications including the identification of peptide ligands by receptors, the mapping of substrate sites for enzymes, and the creation of antibody peptide libraries. These applications are reviewed elsewhere . Inovirus display technology has also been used for epitope mapping and vaccine design purposes. RPLs can be used to characterize the epitope of an antibody of interest through biopanning with a monoclonal antibody of interest, which can result in the isolation of mimotopes; these are oligopeptides that mimic the native antibody epitope. The selected recombinant inoviruses that carry mimotopes can then be isolated in order to determine the DNA and amino acid sequence of the displayed oligopeptides.", "RPLs can be used to characterize the epitope of an antibody of interest through biopanning with a monoclonal antibody of interest, which can result in the isolation of mimotopes; these are oligopeptides that mimic the native antibody epitope. The selected recombinant inoviruses that carry mimotopes can then be isolated in order to determine the DNA and amino acid sequence of the displayed oligopeptides. DNA sequencing of such inserts as well as structure prediction analysis can potentially identify the previously unknown target of an antibody. Besides antibody characterization, inoculation of recombinant inoviruses isolated through this approach can potentially be used as vaccine carriers. For example, if a neutralizing antibody against a pathogen is used to screen an RPL, the selected peptides fused to inoviruses would mimic the original antibody epitope. Vaccination of animals with these inoviruses could ultimately induce the production of similar antibodies by the vaccinated individual, offering protection from infection against the pathogen.", "For example, if a neutralizing antibody against a pathogen is used to screen an RPL, the selected peptides fused to inoviruses would mimic the original antibody epitope. Vaccination of animals with these inoviruses could ultimately induce the production of similar antibodies by the vaccinated individual, offering protection from infection against the pathogen. Inovirus display technology has been successfully applied in the development of vaccines against various pathogens Tables 1 and 2 . The potential for inovirus display technology to facilitate the mapping of antibody epitopes is of great importance, especially in the case of HIV-1. Epitopes of broadly neutralizing monoclonal anti-HIV-1 antibodies could be rare, vulnerable spots on the surface of a frequently mutating virus such as HIV-1 and therefore, their identification and further study could lead to new drug therapies or vaccine targets. This review focuses particularly on the applications of inovirus display technology that utilizes capsid proteins gp3 and gp8, as those have been used in vaccine development.", "Epitopes of broadly neutralizing monoclonal anti-HIV-1 antibodies could be rare, vulnerable spots on the surface of a frequently mutating virus such as HIV-1 and therefore, their identification and further study could lead to new drug therapies or vaccine targets. This review focuses particularly on the applications of inovirus display technology that utilizes capsid proteins gp3 and gp8, as those have been used in vaccine development. Schematic representations of antigen display on the surface of Ff inovirus-associated vectors IAVs . Foreign antigens are shown as red spheres. The designation on the left denotes the inoviral gene, which can be genetically modified to express an antigen on the outer architecture of the virion. IAVs that contain both the wild type and antigen display capsid proteins are designated by \"m\" which indicates that the virion is a mosaic.", "The designation on the left denotes the inoviral gene, which can be genetically modified to express an antigen on the outer architecture of the virion. IAVs that contain both the wild type and antigen display capsid proteins are designated by \"m\" which indicates that the virion is a mosaic. Each Ff virion contains about 2700 copies of major capsid protein gp8, and five copies of each of the minor capsid proteins, gp3, gp6, gp7 and gp9. inovirus-associated vector bottom showing the major coat protein gp8 subunits arranged with a combined five-fold rotation axis and an approximate two-fold screw axis . Right, the corresponding surface lattices, identical to those previously published . The lattice diagrams show the relative position of each gp8 subunit on the outer virion surface.", "Right, the corresponding surface lattices, identical to those previously published . The lattice diagrams show the relative position of each gp8 subunit on the outer virion surface. The five gp8 subunits of each of the two interlocking pentamers constituting the helical symmetry of the virion are indicated by blue and green dots respectively. The relative virion surface area about 1400 Å 2 associated with each gp8 subunit is marked in yellow. The virion perimetrical azimuthial distance is calculated based on a virion diameter of about 65 Å. The displayed antigens, represented by red spheres, are arranged on the surface of the Ff.g8 inovirus-associated vector according to helical symmetry of the virion outer architecture bottom .", "The virion perimetrical azimuthial distance is calculated based on a virion diameter of about 65 Å. The displayed antigens, represented by red spheres, are arranged on the surface of the Ff.g8 inovirus-associated vector according to helical symmetry of the virion outer architecture bottom . IAVs are effective vaccine carriers and, as shown in Tables 1 and 2 , they have been used successfully in numerous vaccine development studies. They have been utilized in the development of vaccines against a wide variety of viral, protozoan and worm parasites and also against non-infectious diseases like Alzheimer and various types of cancer. All these attempts can be divided in two main sub-categories. The first sub-category, inovirus display technology was used to screen RPLs with monoclonal antibodies and then to select the immunogenic peptides of interest.", "All these attempts can be divided in two main sub-categories. The first sub-category, inovirus display technology was used to screen RPLs with monoclonal antibodies and then to select the immunogenic peptides of interest. The selected peptide was used as a vaccine in its soluble form or in conjugation with carrier proteins 55,66,70,81-84, 86-88,90-92,98 . In the second sub-category, similar to the first sub-category, inovirus display technology has been used for epitope mapping and isolation, but in this case, inoviruses were also used as the vaccine carriers for the immunogenic peptides 56, 57, . In contrast to IAVs, soluble peptides have the disadvantage of being less stable than the same peptides fused to inoviral particles. Soluble peptides have a flexible 3D structure and thus, do not always retain the 3D structure of the desirable epitope.", "In contrast to IAVs, soluble peptides have the disadvantage of being less stable than the same peptides fused to inoviral particles. Soluble peptides have a flexible 3D structure and thus, do not always retain the 3D structure of the desirable epitope. As a result, soluble peptides, unlike inovirus-bound peptides, are less capable of inducing the production of the desirable antibodies . Additionally, the inoviral vectors displaying peptides are highly immunogenic. Their high immunogenicity is reinforced by the ability of inoviruses to display multiple copies of peptides on their surface. Additionally, inoviruses are known for their structural simplicity, which allows the immune system to focus selectively on the displayed peptides and not on the viral carrier .", "Their high immunogenicity is reinforced by the ability of inoviruses to display multiple copies of peptides on their surface. Additionally, inoviruses are known for their structural simplicity, which allows the immune system to focus selectively on the displayed peptides and not on the viral carrier . Furthermore, since inoviruses can replicate in E. coli cultures, the cost of vaccine production is low. In summary, IAVs can be used as efficient and cost effective vaccine carriers. A large number of research studies have focused on the application of inovirus-based vaccines against infectious diseases. A common approach in many of these efforts was to vaccinate animals with inoviruses and to then challenge them with specific pathogens, in order to assess the level of protection against the pathogens.", "A large number of research studies have focused on the application of inovirus-based vaccines against infectious diseases. A common approach in many of these efforts was to vaccinate animals with inoviruses and to then challenge them with specific pathogens, in order to assess the level of protection against the pathogens. Three of these studies focused on immunization against viral parasites. In 1997, Bastien et al. fused a 15-mer linear epitope of Human Respiratory Syncytial Virus RSV glycoprotein G on inovirus gp3 and used the recombinant inovirus to vaccinate mice . This resulted in a specific humoral response, with the vaccinated animals having complete protection from challenge with the RSV virus . In 2000, Grabowska et al.", "This resulted in a specific humoral response, with the vaccinated animals having complete protection from challenge with the RSV virus . In 2000, Grabowska et al. used monoclonal antibodies against Herpes Simplex Virus type 2 HSV-2 glycoprotein G2 to screen 15-mer RPLs . The selected recombinant inoviruses were used to vaccinate mice, resulting in a specific humoral response. A high survival rate of vaccinated mice after challenge with a lethal dose of the virus was observed, and the level of protection was proportional to the dose of the inoviral vaccine . Additionally, in 2000, Yu et al. used monoclonal antibodies against the surface glycoprotein of Neurotropic Murine Coronavirus to screen various RPLs .", "Additionally, in 2000, Yu et al. used monoclonal antibodies against the surface glycoprotein of Neurotropic Murine Coronavirus to screen various RPLs . A selected clone displaying a 13-mer peptide induced a humoral immune response but no cellular response in mice. Even so, after lethal virus challenge, three out of six mice survived. . Besides viral infections, inoviral vaccine research has also been applied for systemic candidiasis, a fungal infection caused by Candida albicans. In two separate studies, a specific six amino acid peptide epitope of the fungal heat shock protein 90 was displayed as a fusion with the inoviral coat protein gp8 .", "Besides viral infections, inoviral vaccine research has also been applied for systemic candidiasis, a fungal infection caused by Candida albicans. In two separate studies, a specific six amino acid peptide epitope of the fungal heat shock protein 90 was displayed as a fusion with the inoviral coat protein gp8 . After infection with the parasite, mice immunized with the recombinant inoviruses had fewer colony forming units of Candida albicans in the kidney and a longer lifespan . The use of inovirus as a vaccine carrier was particularly successful against Taenia solium, a parasitic worm that uses pigs as intermediate hosts and causes neurocysticercosis, a parasitic disease of the central nervous system, in humans . In 2004, Manoutsarian et al. fused four antigenic peptides GK1, KETc1, KETc7, KETc12 to inoviruses and the cocktail of recombinant inoviruses was used to vaccinate pigs; as a result, a specific cellular response was induced.", "In 2004, Manoutsarian et al. fused four antigenic peptides GK1, KETc1, KETc7, KETc12 to inoviruses and the cocktail of recombinant inoviruses was used to vaccinate pigs; as a result, a specific cellular response was induced. Vaccination of pigs protected them against challenge with the pathogen: 1/3 of pigs were totally protected and 2/3 had reduced number of cysticerci . Based on these results, large-scale vaccination of 1047 rural pigs in 16 villages of central Mexico was conducted in 2008. The immunization was successful since the vaccine conferred significant protection against the parasite. Furthermore, this inovirus-based vaccine was more economical compared with to another vaccine made of synthetic peptides.", "The immunization was successful since the vaccine conferred significant protection against the parasite. Furthermore, this inovirus-based vaccine was more economical compared with to another vaccine made of synthetic peptides. The study was particularly important, not only due to the large scale vaccination attempt with inoviruses, but also due to its effectiveness in significantly reducing the number of cysticerci in the vaccinated animals . Efforts were also made to develop vaccines against the worm Schistosoma japonicum. In 2004, Tang et al. screened a 12-mer RPL with polyclonal serum from infected mice . The selected recombinant inoviruses induced a specific humoral response, which conferred partial protection from parasite challenge in the vaccinated mice .", "screened a 12-mer RPL with polyclonal serum from infected mice . The selected recombinant inoviruses induced a specific humoral response, which conferred partial protection from parasite challenge in the vaccinated mice . Following this study, in 2006, Wang et al. used the monoclonal antibody SSj14, which targets the parasite to screen a 12-mer RPL . The recombinant inoviruses induced both humoral and cellular responses that significantly protected the vaccinated mice against the worm infection . Additionally, in 2006, Wu et al. used polyclonal serum from infected rabbits to screen a 12-mer RPL . A humoral response was induced in the vaccinated mice, which conferred partial protection from the parasite .", "used polyclonal serum from infected rabbits to screen a 12-mer RPL . A humoral response was induced in the vaccinated mice, which conferred partial protection from the parasite . In 2008, Villa-Mancera et al., produced a vaccine against Fasciola hepatica, by screening a previously constructed 12-mer inovirus RLP with an anti-cathepsin L monoclonal antibody . Although immunization of sheep with the selected recombinant inoviruses induced a weak, specific humoral response after challenge with the parasite, the vaccinated animals had a remarkable reduction in worm burden compared to controls . An inovirus-based vaccine has been constructed against the parasitic worm Trichinella spiralis. In this case, Gu et al.", "An inovirus-based vaccine has been constructed against the parasitic worm Trichinella spiralis. In this case, Gu et al. used a monoclonal antibody against rTs87 antigen to screen a 12-mer RPL . As a result, a humoral response was induced and the vaccinated mice gained partial protection after challenge against the parasite . In summary, the results of the above studies show that the construction of an efficient inovirus-based vaccine that confers protection to the vaccinated animals against the infectious pathogen is achievable through the induction of humoral or cellular immune response or both. As previously mentioned, inovirus display technology has also been used for the design of vaccines against non-infectious diseases and in some cases, the capability of the vaccine to limit the progression of the disease was evaluated.", "In summary, the results of the above studies show that the construction of an efficient inovirus-based vaccine that confers protection to the vaccinated animals against the infectious pathogen is achievable through the induction of humoral or cellular immune response or both. As previously mentioned, inovirus display technology has also been used for the design of vaccines against non-infectious diseases and in some cases, the capability of the vaccine to limit the progression of the disease was evaluated. In 2005, Fang et al. displayed an epitope of the Melanoma Antigen A1 MAGE A1 in the surface of inovirus fused on gp8 . This resulted in an induction of cellular immune response against the melanoma tumor and in the significant inhibition of tumor growth in the vaccinated mice. In addition, there was an important increase in the survival rate of vaccinated animals .", "This resulted in an induction of cellular immune response against the melanoma tumor and in the significant inhibition of tumor growth in the vaccinated mice. In addition, there was an important increase in the survival rate of vaccinated animals . Similar results were obtained in 2002 by Wu et al. in an effort to design a vaccine against murine mastocytoma P815 . An epitope of the P1A tumor antigen was fused to the inoviral surface and the vaccinated mice gained significant protection against tumor growth. Moreover, there was a significant increase in survival rate due to an anti-tumor cellular response that was induced .", "An epitope of the P1A tumor antigen was fused to the inoviral surface and the vaccinated mice gained significant protection against tumor growth. Moreover, there was a significant increase in survival rate due to an anti-tumor cellular response that was induced . Some important efforts have also been made for the design of an inovirus-based vaccine against Alzheimer's disease. The main target of these vaccines was the induction of antibodies against β-amyloid plaques. In these studies, the antigenic epitope that was displayed in the inoviral surface was the epitope EGFR, which consists of the four amino acids E, G, F and R and it is part of the β-amyloid peptide. In mice immunized with recombinant inoviruses, the researchers observed a reduction in β-amyloid plaque burden and a specific humoral response .", "In these studies, the antigenic epitope that was displayed in the inoviral surface was the epitope EGFR, which consists of the four amino acids E, G, F and R and it is part of the β-amyloid peptide. In mice immunized with recombinant inoviruses, the researchers observed a reduction in β-amyloid plaque burden and a specific humoral response . Collectively, these studies show that it is possible to protect vaccinated animals against disease progression, thus alluding to the promising use of such vaccines against non-infectious diseases in humans. In summary, the utilization of inoviral vectors for vaccine development against infectious and non-infectious non-HIV-1 diseases has produced significant and promising results. First, in the majority of cases, the vaccine was successful since it induced specific humoral or cellular response, or both. Furthermore, in many cases, there was an attempt to evaluate the efficacy of the vaccine after challenge against the pathogen in vaccinated animals.", "First, in the majority of cases, the vaccine was successful since it induced specific humoral or cellular response, or both. Furthermore, in many cases, there was an attempt to evaluate the efficacy of the vaccine after challenge against the pathogen in vaccinated animals. In all cases, the vaccine could provide partial, significant or even complete protection against the pathogen. This established the effectiveness of the use of inoviruses as vaccine vectors. Our knowledge of HIV-1 neutralization epitopes initiated from the isolation of several neutralizing monoclonal antibodies 2F5, 4E10, b12 and 2G12 that were described between 1993 and 1994 . Thus far, neutralizing monoclonal antibodies have been found to target four major epitopes on the HIV-1 envelope gp41 and gp120 glycoproteins .", "Our knowledge of HIV-1 neutralization epitopes initiated from the isolation of several neutralizing monoclonal antibodies 2F5, 4E10, b12 and 2G12 that were described between 1993 and 1994 . Thus far, neutralizing monoclonal antibodies have been found to target four major epitopes on the HIV-1 envelope gp41 and gp120 glycoproteins . These monoclonal antibodies target the MPER epitope of gp41 monoclonal antibodies 2F5, 4E10, M66.6, CAP206-CH12 and 10e8 ; the V1V2-glycan of gp120 PG9, PG16, CH01-04 and PGT 141-145 ; the glycan dependent site of the gp120 V3 loop PGT121-123, PGT125-131 and PGT135-137 ; and the CD4-binding site b12, HJ16, CH103-106, VRC01-03, VRC-PG04, VRC-PG04b, VRC-CH30-34, 3BNC117, 3BNC60, NIH45-46, 12A12, 12A21, 8ANC131, 8ANC134, INC9 and IB2530 102, 114, . Monoclonal antibody 2G12 targets the surface glycans on the outer domain of gp120 that is distinct from the four major epitope target sites described above . The inovirus display technology has also been applied in vaccination strategies against HIV-1 . However, unlike the successful development of vaccines against non-HIV-1 parasites, these efforts failed.", "The inovirus display technology has also been applied in vaccination strategies against HIV-1 . However, unlike the successful development of vaccines against non-HIV-1 parasites, these efforts failed. In all published studies see Table 1 , the HIV-1 inovirus-display vaccines were made utilizing the broadly neutralizing antibodies 2F5, 2G12 and b12 . The first study for the construction of a vaccine against HIV-1 using inovirus display technology was performed in 1993 by Keller et al., using the 447-52D monoclonal antibody to screen a 15-mer RPL . Vaccination of selected recombinant inoviruses in rabbits resulted in the induction of type-specific neutralizing antibodies . A few years later, in 2001, Zwick et al.", "Vaccination of selected recombinant inoviruses in rabbits resulted in the induction of type-specific neutralizing antibodies . A few years later, in 2001, Zwick et al. used b12 monoclonal antibody to screen a variety of linear and constrained RPLs . However, the vaccination in mice and rabbits with the selected recombinant inoviruses did not result in the production of b12-like antibodies at detectable levels . The same antibody was used in 2005 by Dorgham et al. , in order to screen a 15-mer RPL, and the selected mimotopes were fused to the capsid of inoviruses which were then used to vaccinate mice . The induced antibodies could bind gp160 but they did not have neutralizing potency .", ", in order to screen a 15-mer RPL, and the selected mimotopes were fused to the capsid of inoviruses which were then used to vaccinate mice . The induced antibodies could bind gp160 but they did not have neutralizing potency . In another study in 2007, Wilkinson et al. screened a 9-mer and a constrained 10-mer library with antibody 5145A . This was the only case in which the selected mimotopes were fused in to small heat shock protein HSP of the archeaon Methanococcus jannaschii as a carrier protein. Following vaccination of HSP-mimotopes in rabbits, anti-gp120 antibodies without neutralizing potency were produced . The 2G12 antibody was used for the first time in 2008 by Menendez et al.", "Following vaccination of HSP-mimotopes in rabbits, anti-gp120 antibodies without neutralizing potency were produced . The 2G12 antibody was used for the first time in 2008 by Menendez et al. for the screening of a variety of linear and constrained RPLs . Nevertheless, vaccination of selected inoviruses in rabbits induced the production of antibodies that could not bind to gp120 . More recently, in 2011, Rodriguez et al. used the 2F5 antibody to screen a 12-mer and a constrained 7-mer RPL . Vaccination in mice and rabbits led to the production of non-neutralizing antibodies . In most of these studies, the use of inoviral vectors resulted in the induction of a specific humoral response.", "Vaccination in mice and rabbits led to the production of non-neutralizing antibodies . In most of these studies, the use of inoviral vectors resulted in the induction of a specific humoral response. However, the sera of the vaccinated animals did not have broadly neutralizing ability. Besides monoclonal antibodies, polyclonal sera from HIV-1-infected patients were also used for the screening RPLs Table 1 51, 53, 63 . This approach carries a degree of uncertainty, since it is based on the premise that the polyclonal sera will contain at least one broadly neutralizing monoclonal anti-HIV-1 antibody, meaning that a new neutralizing epitope against it can be isolated from the RPL. It is suggested that long-term non-progressors HIV-1-infected patients who remain asymptomatic for a long time are more likely to produce neutralizing antibodies in comparison to AIDS patients, and it is suggested that these neutralizing antibodies in the serum of long-term non-progressors may contribute to the control of viral load .", "This approach carries a degree of uncertainty, since it is based on the premise that the polyclonal sera will contain at least one broadly neutralizing monoclonal anti-HIV-1 antibody, meaning that a new neutralizing epitope against it can be isolated from the RPL. It is suggested that long-term non-progressors HIV-1-infected patients who remain asymptomatic for a long time are more likely to produce neutralizing antibodies in comparison to AIDS patients, and it is suggested that these neutralizing antibodies in the serum of long-term non-progressors may contribute to the control of viral load . However, this hypothesis has been questioned . Polyclonal serum for the screening of RPLs was used for the first time in 1999 by Scala et al., who screened both linear and constrained 9-mer RPLs . After that, vaccination of selected inoviruses in mice led to the production of neutralizing antibodies. A few years later, in 2007, Rodriguez et al.", "After that, vaccination of selected inoviruses in mice led to the production of neutralizing antibodies. A few years later, in 2007, Rodriguez et al. used polyclonal serum to screen a 7-mer, a 12-mer and a constrained 7-mer RPL . Vaccination in mice induced the production of antibodies that could bind to gp41, but no information was provided about their neutralization potency . Additionally, in 2007, Humbert et al. used polyclonal serum to screen a 7-mer, a 12-mer and a constrained 7-mer RPL . Vaccination of selected recombinant inoviruses in mice induced the production of neutralizing antibodies .", "used polyclonal serum to screen a 7-mer, a 12-mer and a constrained 7-mer RPL . Vaccination of selected recombinant inoviruses in mice induced the production of neutralizing antibodies . The screening of the same RPLs using polyclonal serum from a monkey infected with a Simian-Human Immunodeficiency Virus SHIV was performed by the same group. In this study, vaccination in mice was performed using the prime-boost strategy: DNA vaccine as prime encoding gp160 and a cocktail of recombinant inoviruses as boost. The result was the induction of neutralizing antibodies . In 2013, Gazarian et al. screened a linear 12-mer and constrained 7-mer RPLs using sera from HIV-infected individuals .", "The result was the induction of neutralizing antibodies . In 2013, Gazarian et al. screened a linear 12-mer and constrained 7-mer RPLs using sera from HIV-infected individuals . Vaccination in rabbits resulted in the production of antibodies that bind gp160 . In 2001, non-human primates were used by Chen et al., as an animal model for vaccination with recombinant inoviruses . This group performed screening of a 9-mer and a constrained 9-mer RPL with polyclonal serum isolated from an infected donor. After that, the vaccination of selected inoviruses was performed in rhesus macaques. As a result, sera from the vaccinated macaques exhibited neutralizing activity.", "After that, the vaccination of selected inoviruses was performed in rhesus macaques. As a result, sera from the vaccinated macaques exhibited neutralizing activity. Furthermore, the vaccinated macaques were not protected from infection, but four out of five animals were able to control the viral load after challenge against the pathogenic SHIV89.6PD virus. This was a very important result, which underlines the potential of the method. Additionally, no specific CTL response was detected, implying that the control of viral load was an exclusive result of humoral response . Since the first attempt to induce the production of anti-HIV-1 neutralizing antibodies using inovirus-based vaccines, all efforts to date have not led to the production of broadly neutralizing antibodies in vaccinated animals.", "Additionally, no specific CTL response was detected, implying that the control of viral load was an exclusive result of humoral response . Since the first attempt to induce the production of anti-HIV-1 neutralizing antibodies using inovirus-based vaccines, all efforts to date have not led to the production of broadly neutralizing antibodies in vaccinated animals. This failure could be to a certain extent explained by the usage of the \"old generation\" monoclonal antibodies 2F5, 2G12 and b12 which were shown to demonstrate autoreactivity properties in vitro studies . In 2010, Verkoczy et al. demonstarted in an in vivo study that Pre-B cells expressing 2F5-like antibodies were unable to maturate in mice, suggesting a triggering of immunological tolerance due to the autoreactive properties of the 2F5-like antibody . Collectively, these studies implied that the screening RPLs using 2F5, 2G12 and b12 could result in inoviral-based vaccines that could trigger immunological tolerance in vaccinated animals.", "demonstarted in an in vivo study that Pre-B cells expressing 2F5-like antibodies were unable to maturate in mice, suggesting a triggering of immunological tolerance due to the autoreactive properties of the 2F5-like antibody . Collectively, these studies implied that the screening RPLs using 2F5, 2G12 and b12 could result in inoviral-based vaccines that could trigger immunological tolerance in vaccinated animals. It is important to note, however, that the lack of autoreactivity properties for several of the \"next generation\" broadly neutralizing antibodies 10e8, PG9, PG16, VRC01-03, VRC-PG04, VRC-PG04b, and VRC-CH30-34 could solve the autoreactivity problems encountered by 2F5, 2G12, and b12. Despite the fact that the recent isolation of new broadly neutralizing antibodies against HIV-1 has focused the attention of HIV-1 vaccine development on the induction of a humoral anti-HIV-1 response, recent results underline the importance of a cellular anti-HIV-1 response as well . The experimental results concerning inovirus-based vaccines in non-HIV-1 diseases prove that inoviruses can also induce a strong specific cellular response Tables 1 and 2 ; this property makes them great candidates as vectors for HIV-1 vaccine design. The ability of inoviruses to induce a cellular immune response was first demonstrated in 2000 by DeBerardinis et al.", "The experimental results concerning inovirus-based vaccines in non-HIV-1 diseases prove that inoviruses can also induce a strong specific cellular response Tables 1 and 2 ; this property makes them great candidates as vectors for HIV-1 vaccine design. The ability of inoviruses to induce a cellular immune response was first demonstrated in 2000 by DeBerardinis et al. . In this work, recombinant inoviruses carrying the RT2 epitope and the pep23 epitope of HIV-1 reverse transcriptase in gp8 could induce specific cellular responses in human cell lines in vitro and in mice in vivo against the RT2 peptide. It is interesting that without the pep23 epitope, the cellular response was undetectable. This indicates that the pep23 is a CTL epitope that is necessary for cellular response, possibly because it enables internalization of the recombinant inovirus into the APCs .", "It is interesting that without the pep23 epitope, the cellular response was undetectable. This indicates that the pep23 is a CTL epitope that is necessary for cellular response, possibly because it enables internalization of the recombinant inovirus into the APCs . Therefore, a question arises of whether an epitope fused to the surface of the inovirus can induce a cellular or a humoral response or both. It is suggested that the type of immune response caused by an epitope fused on the surface of the inovirus is dependent on the length and sequence of the peptide . In 2003, Gaubin et al. demonstrated that FITC-labeled fd virions can be internalized in human EBV-B cell lines and the fd virions are successively degraded and targeted both to MHC class I and class II antigen-processing pathways .", "In 2003, Gaubin et al. demonstrated that FITC-labeled fd virions can be internalized in human EBV-B cell lines and the fd virions are successively degraded and targeted both to MHC class I and class II antigen-processing pathways . This was confirmed after endocellular localization of the labeled virions with confocal microscopy. This experiment showed that the inoviruses could be internalized in APCs even without carrying a CTL epitope, but in very low rate. For in vivo experiments however, it is possible that the requirement of a CTL epitope is critical for the induction of a cellular response . In 2011, Sartorius et al.", "For in vivo experiments however, it is possible that the requirement of a CTL epitope is critical for the induction of a cellular response . In 2011, Sartorius et al. showed that a hybrid fd virion with the anti-DEC-205 scFv antibody fragment fused on gp3 and the OVA257-264 antigenic epitope fused on gp8 can be internalized in human dendritic cells through a specific interaction between the anti-DEC205 scFv and the DEC-205 receptor . In addition, inoculation of mice with the hybrid virions induced a specific cellular response against the OVA257-264 epitope . This important characteristic of inoviral vectors to induce a cellular response was reported in only two studies aimed at developing a HIV-1 inoviral vaccine, possibly due to the complexity of detecting a cellular response and also because it is a labor intensive and time-consuming process. In 2001, Chen et al.", "This important characteristic of inoviral vectors to induce a cellular response was reported in only two studies aimed at developing a HIV-1 inoviral vaccine, possibly due to the complexity of detecting a cellular response and also because it is a labor intensive and time-consuming process. In 2001, Chen et al. attempted to detect a cellular response, but such a response was not induced in that experiment . A more recent research study related to HIV-1, which clearly demonstrates the ability of inoviruses to induce strong cellular response, was performed in 2009 by Pedroza-Roldan et al. . In this effort, an immunodominant CTL epitope of the V3 loop of gp120 residues 311-320 was expressed as fusion to gp8 of an M13 inovirus.", ". In this effort, an immunodominant CTL epitope of the V3 loop of gp120 residues 311-320 was expressed as fusion to gp8 of an M13 inovirus. A random peptide library was created by inserting mutations in certain positions of this epitope. A cocktail of inoviruses carrying the V3 loop epitope or variations of this epitope were used for the vaccination of mice and, as a result, a CTL response was induced. The most important result of this study was that the immunization induced long-lasting memory T-cell responses, which were detected seven months after a single immunization . The same vaccination also induced a strong humoral response, since the sera of vaccinated mice could neutralize five out of ten pseudoviruses from a panel .", "The most important result of this study was that the immunization induced long-lasting memory T-cell responses, which were detected seven months after a single immunization . The same vaccination also induced a strong humoral response, since the sera of vaccinated mice could neutralize five out of ten pseudoviruses from a panel . All the above experiments clearly show that the inoviruses are capable of inducing a specific cellular immune response: the ability of the inoviral vectors to induce both arms of adaptive immunity is unique and it could prove to be valuable in the development of a successful HIV-1 vaccine. In the general field of HIV-1 vaccine design, all studies for the production of anti-HIV-1 broadly neutralizing antibodies through vaccination with either soluble peptides or viral vectors have been unsuccessful. For this reason, some efforts have been directed to the induction of cellular immune response . In recent years, in HIV-1 vaccine phase I and phase II clinical trials, adenoviral vectors have been used in order induce a cellular immune response in HIV-1 vaccinated individuals .", "For this reason, some efforts have been directed to the induction of cellular immune response . In recent years, in HIV-1 vaccine phase I and phase II clinical trials, adenoviral vectors have been used in order induce a cellular immune response in HIV-1 vaccinated individuals . However, there are concerns about the safety of these viruses. In 2007, the large-scale phase IIB Merck trial was abruptly terminated because there was evidence that the individuals vaccinated with adenovirus rAd5 vector expressing gag, pol and nef became more vulnerable to HIV-1 infection in comparison to controls. It was suggested that the group with the increased risk of being infected with HIV-1 consisted of individuals who were Ad5 seropositive . Other eukaryotic viruses that infect other species and do not replicate in human cells were also tested as candidates HIV vaccine carriers and in theory are safer.", "It was suggested that the group with the increased risk of being infected with HIV-1 consisted of individuals who were Ad5 seropositive . Other eukaryotic viruses that infect other species and do not replicate in human cells were also tested as candidates HIV vaccine carriers and in theory are safer. For example, the canarypox vector was used in the RV144 phase III clinical trial, the only clinical trial that had positive results to date, offering partial protection 31% to vaccinated individuals in comparison with control . Apart from safety reasons, the use of adenovirus-based vectors has not been protective. The recent HVTV 505 phase IIB trial that used adenovirus rAd5 as a boost and DNA as prime for vaccination of 2504 human volunteers was abandoned as futile . Recently, a rhesus macaque cytomegalovirus RhCMV vector successfully induced a persisting CTL response in rhesus macaques that strongly protected the vaccinated animals from challenge against the pathogenic SIVmac239 strain.", "The recent HVTV 505 phase IIB trial that used adenovirus rAd5 as a boost and DNA as prime for vaccination of 2504 human volunteers was abandoned as futile . Recently, a rhesus macaque cytomegalovirus RhCMV vector successfully induced a persisting CTL response in rhesus macaques that strongly protected the vaccinated animals from challenge against the pathogenic SIVmac239 strain. Importantly, this study, 50% of the vaccinated animals reduced the viral load to undetectable levels . However, the design of a human version of this CMV vector could impose safety risks, since the human CMV is a persistent and pathogenic human virus . Therefore, various types of eukaryotic viral vectors are currently under investigation. These are reviewed elsewhere .", "Therefore, various types of eukaryotic viral vectors are currently under investigation. These are reviewed elsewhere . However, the use of a eukaryotic virus is accompanied by serious safety concerns. As an alternative, the use of prokaryotic viruses such as inoviruses, may be utilized which have a decisively lower safety risk to humans. Even if inoviruses could infect a eukaryotic cell, the assembly of the new prokaryotic virions cannot take place without the specific conditions that exist in the inter-membrane area of the E. coli and without the presence of the specific E. coli enzyme leader peptidase that does not exist in human cells . Furthermore, there was a phase I case study in 2006 where fd inoviruses were intravenously infused in humans for purposes unrelated to vaccination , causing no side effects or even allergic reactions in any of the eight volunteers.", "Even if inoviruses could infect a eukaryotic cell, the assembly of the new prokaryotic virions cannot take place without the specific conditions that exist in the inter-membrane area of the E. coli and without the presence of the specific E. coli enzyme leader peptidase that does not exist in human cells . Furthermore, there was a phase I case study in 2006 where fd inoviruses were intravenously infused in humans for purposes unrelated to vaccination , causing no side effects or even allergic reactions in any of the eight volunteers. To our knowledge, this is the only case where inoviruses were infused in humans . Therefore, in contrast to other viral vectors, the use of inoviruses does not impose a major safety risk to humans. This characteristic of inoviral vectors along with their capability to trigger both cellular and humoral immune responses makes them an attractive option as vaccine vectors. During the last two decades, inoviral vectors have been used in the development of vaccines against various infectious parasites and against non-infectious diseases like cancer and Alzheimer's with promising results.", "This characteristic of inoviral vectors along with their capability to trigger both cellular and humoral immune responses makes them an attractive option as vaccine vectors. During the last two decades, inoviral vectors have been used in the development of vaccines against various infectious parasites and against non-infectious diseases like cancer and Alzheimer's with promising results. While the applications of inovirus display technology in vaccine design against non-HIV-1 diseases have been mostly successful, the design of a HIV-1 vaccine development has so far been disappointing. A major obstacle has been the use of neutralizing monoclonal antibodies plagued with autoreactivity properties. Screening of RPLs with these antibodies resulted in the isolation of peptides that, as vaccine antigens, were unsuccessful in inducing a specific humoral response that would produce neutralizing antibodies. The recent isolation of antibodies such as VRC01 and 10E8 with more neutralizing breadth and potency without autoreactivity properties than the previously utilized antibodies may overcome this obstacle .", "Screening of RPLs with these antibodies resulted in the isolation of peptides that, as vaccine antigens, were unsuccessful in inducing a specific humoral response that would produce neutralizing antibodies. The recent isolation of antibodies such as VRC01 and 10E8 with more neutralizing breadth and potency without autoreactivity properties than the previously utilized antibodies may overcome this obstacle . Particularly, the induction of VRC01-like and 10E8-like antibodies could be a feasible target, since these antibodies also seem to be produced from a significant percentage of the HIV-1-infected population . While humoral responses have been well documented, cellular responses have not been assessed in most studies for the design of a HIV-1 vaccine using inoviruses. The only study in which a cellular anti-HIV-1 response was detected also reported a successful induction of a long-lasting memory CTL response seven months after a single vaccination in mice with inoviral particles , demonstrating that the induction of cellular immunity against HIV-1 using inoviruses is feasible. IAVs are advantageous in that they can induce both arms of adaptive immunity.", "The only study in which a cellular anti-HIV-1 response was detected also reported a successful induction of a long-lasting memory CTL response seven months after a single vaccination in mice with inoviral particles , demonstrating that the induction of cellular immunity against HIV-1 using inoviruses is feasible. IAVs are advantageous in that they can induce both arms of adaptive immunity. This finding could therefore be of importance in future efforts for the design of a HIV-1 vaccine. Moreover, IAVs have unique characteristics compared to other viral vectors: they are stable, they can display a peptide in multiple from few to thousands copies on their surface and such constructs are very immunogenic without the use of an adjuvant. In addition, IAVs allow the immune system to focus on a specific epitope of interest instead of the whole protein, which is of great importance, since an important aspect for successful HIV-1 vaccine design is to focus on the induction of neutralizing antibodies against the specific neutralizing epitopes while at the same time avoiding the induction of ineffective antibodies against the numerous non-neutralizing epitopes of the HIV-1 glycoproteins, which act as decoys for the immune system. Ideally, an effective HIV-1 vaccine should be able to stimulate both humoral and cellular immune responses.", "In addition, IAVs allow the immune system to focus on a specific epitope of interest instead of the whole protein, which is of great importance, since an important aspect for successful HIV-1 vaccine design is to focus on the induction of neutralizing antibodies against the specific neutralizing epitopes while at the same time avoiding the induction of ineffective antibodies against the numerous non-neutralizing epitopes of the HIV-1 glycoproteins, which act as decoys for the immune system. Ideally, an effective HIV-1 vaccine should be able to stimulate both humoral and cellular immune responses. Recently, adenoviral vectors were tested in clinical trials as HIV-1 vaccine carriers in order to induce cellular immunity, but they were shown to impose serious health risks for humans. On the other hand, IAVs are able to induce cellular immunity and at the same time they have been demonstrated to be safe for administration in animals and humans. These characteristics of IAVs, conferred by their structural and biological properties, make them effective antigen display vectors that can induce strong and specific humoral and cellular immune responses against the displayed antigen. These properties of IAVs along with newly discovered broadly neutralizing anti-HIV-1 antibodies, pave the way for the development of an effective HIV-1 vaccine." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with pgsA-CTA1-sM2/L. casei or without pgsA-sM2/L. casei cholera toxin subunit A1 CTA1 on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G IgG and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD.", "However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD. of A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird /Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/Chicken/Korea/116/2004 H9N2 viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses .", "These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text: Vaccination remains most economical and effective means against respiratory diseases caused by influenza viruses . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period .", "However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 M2 is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections.", "In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli E. coli expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 and peptides encoding M2e , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein , keyhole limpet hemocyanin and flagellin . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B .", "Mice immunized mucosally with an M2 or virus like particles VLPs adjuvanted with cholera toxin CT demonstrated better protection compared to mice subjected to parenteral immunization . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B . E. coli expressing cholera toxin subunit A1 CTA1 fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied .", "Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria LAB presenting influenza virus antigens have been studied . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane.", "Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses no trans-membrane domain with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages.", "Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice 5 weeks old were purchased from Samtako Seoul, Korea and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level BSL -2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group.", "Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 3 mice for lung histopathology and immunohistochemistry , and the last contained 11 mice per group 3 mice for CTL response . Concentrations of recombinant L. casei were determined by colony forming units CFU . In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L.", "In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis.", "Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea .", "The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06 H5N1 , A/Puerto Rico/8/34 H1N1 , A/Aquatic bird/Korea/W81/2005 H5N2 , A/Aquatic bird/Korea/W44/2005 H7N3 , and A/ Chicken/Korea/116/2004 H9N2 used in this study were kindly provided by Dr. Young-Ki Choi College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea . All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses MLD 50 were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival.", "Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses.", "After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively.", "casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation Daejeon, South Korea . A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L.", "The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39.", "The polymerase chain reaction PCR was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector Invitrogen, Seoul, Korea . Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2.", "The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing Solgent, Seoul, Korea . After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours.", "casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline PBS . Bacterial lyses were performed by sonication and centrifuged at 12,0006g for 20 minutes at 4uC. Cell wall and cytoplasmic fractions were separated by centrifugation at 25,0006g at 4uC for 2 hours. Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously.", "Pellets cell wall were resuspended in 100 ml of 1% sarcosol containing 1 mM phenylmethylsulfonyl fluoride PMSF, Sigma-Aldrich, St. Louis, USA as a protease inhibitor. Fractions were analyzed by western blotting, as described previously. For the immune detection of fusion proteins, the membranes were probed with rabbit anti-cholera toxin 1:2000, Abcam, UK , rabbit anti-pgsA 1:1000 and rabbit anti-M2 1:1000 antibodies. The rabbit anti-pgsA and rabbit anti-M2 antibodies used in this experiment were generated by the i.m. inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP .", "inoculation of KLH-conjugated pgsA or M2 peptide in rabbit, respectively, two times at 2 weeks-interval. The membranes were reacted with a 1:10,000 dilution of anti-rabbit immunoglobulin G conjugated with horseradish peroxidase IgG HRP . Finally, the target proteins were detected using the WEST-ZOL plus Western Blot Detection System iNtRON Biotechnology, Gyeonggi-do, Korea and visualized by enhanced chemiluminescence ECL . To investigate the expression of sM2 or CTA1-sM2 on the surface of L. casei, recombinant L. casei were grown in 30uC for 48 hours in the MRS broth. Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours.", "Bacteria were harvested by centrifugation at 5,0006g for 10 minutes at 4uC, washed three times with sterile phosphate-buffered saline containing 0.01% Tween-20 PBST and probed with polyclonal rabbit anti-M2 or rabbit anti-CT antibody overnight. Following another washing, the cells were treated with fluorescein isothiocyanate FITC conjugated anti-rabbit IgG antibodies Burlingame, CA, USA for 2 hours. Finally, 10,000 cells were analyzed by flow cytometry Becton Dickinson, Oxnard, CA, USA . For the immunofluorescence, cells were prepared under the same condition described for the flow cytometry. The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope.", "The pgsA/L. casei was used as a negative control and Immunofluoresence analysis was examined using a Carl Zeiss Axioskop 2 fluorescence microscope. ELISA Antibody titers were measured by enzyme-linked immunosorbent assay ELISA using serum or mucosal samples from vaccinated mice. First, 96-well immunosorbent plates Nunc were incubated with 300 ng/well purified sM2 or CTA1 proteins at 4uC overnight. The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC.", "The recombinant sM2 and CTA1 proteins used in this study were purified from E. coli. Next, the wells were blocked with 10% skim milk for 2 hours in RT, washed five times with PBST, treated with diluted serum samples 1:200 in triplicate for detecting IgG and undiluted tissue homogenized supernatant for detecting local IgA and incubated for 2 hours at 37uC. After washing three times, goat anti-mouse IgG HRP 1:1000, sigma or anti-mouse IgA was added to each well and incubated for an additional 2 hours at 37uC. Following another round of washing, the plates were reacted with the substrate solution containing tetramethylbenzidine and H 2 O 2 and allowed to precede the reaction for 10 minutes. After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously .", "After adding the stop solution 2N-H 2 SO 4 , the optical density OD was measured at 450 nm using an ELISA autoreader Molecular devices . The development and counting of cytokines were performed by ELISPOTs, as described previously . Briefly, the day before the isolation of splenocytes, ELISPOT 96-well plates were coated with monoclonal anti-mouse IFN-c and IL-4 capture antibodies 5 mg/ml in PBS and incubated at 4uC overnight. The plates were washed with PBS, and 200 ml/well of blocking solution containing complete RPMI 1640 medium and 10% fetal bovine serum, was added Invitrogen, Carlsbad, CA, USA and incubated for 2 hours in RT. Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 .", "Spleens from the vaccinated mice were isolated aseptically and added at 5610 4 cells/well in media containing sM2 protein, M2 peptide SLLTEVETPTRNGWECKCSD 1 mg/well , only medium negative control , or 5 mg/ml phytohemagglutinin positive control, Invitrogen, Carlsbad, CA, USA . After adding cells and stimulators, the plates were incubated for 24 hours at 37uC with 5% CO 2 . The plates were sequentially treated with biotinylated anti-mouse IFN-c and IL-4 antibodies, streptavidinhorseradish peroxidase, and substrate solution. Finally, the spots were counted using an ImmunoScan Entry analyzer Cellular Technology, Shaker Heights, USA . The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA .", "The lungs were collected aseptically, and virus titers were determined by 50% tissue culture infectious dose TCID 50 , as described previously . Briefly, lung tissues were homogenized in 500 ml of PBS containing antibiotics penicillin, and streptomycin and antimycotics Fungizone compounds Gibco, Grand Island, NY, USA . Mechanically homogenized lung samples were centrifuged 15 minutes, 12,0006g and 4uC to remove the cellular debris before their storage at 280uC. MDCK cells were inoculated with a 10-fold serially diluted sample and incubated at 37uC in a humid atmosphere of 5% CO 2 for an hour. After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test.", "After absorption, the media was removed, and overlay medium containing L-1-tosylamido-2-phenylethyl chloromethyl ketone TPCK trypsin Thermo Fisher Scientific, Rockford, USA was added to the infected cells and incubated for 72 hours. Viral cytopathic effects were observed daily, and the titers were determined by the HA test. The viral titer of each sample was expressed as 50% tissue infected doses using the Reed-Muench method . For histopathology, lung tissues were collected at 5 dpi from ether narcosis-anesthetized mice. Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described .", "Tissues were immediately fixed in 10% formalin containing neutral buffer, embedded in paraffin wax, sectioned at 4-6 mm thickness using a microtome machine, mounted onto slides, and stained with eosin stain. Histopathological changes were examined by light microscopy, as previously described . Furthermore, slides were stained using an immunoperoxidase method with an antibody rabbit anti-M2, 1:500 directed against the matrix protein-2 of influenza A virus. A Goat-anti-rabbit IgG HRP 1:2000, Sigma-Aldrich, St. Louis, USA was used as the secondary antibody for the detection of virus infected cells in respective tissues . Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments.", "Data are presented as the means 6 standard deviations S.D. and are representative of at least three independent experiments. Differences between groups were analyzed by analysis of variance ANOVA , and means were compared by Student's t-test. P-values less than 0.05 were regarded as significant. Results for percent initial body weight were also compared by using Student's t test. Comparison of survival was done by log-rank test using GraphPad Prism 6 version. The pgsA-expressing vector was used to construct plasmids containing the highly conserved consensus sM2 gene, with pgsA-CTA1-sM2 or without pgsA-sM2 the cholera toxin subunit A1 CTA1, Fig. 1A . Plasmids were transformed into L. casei cells.", "1A . Plasmids were transformed into L. casei cells. The expression levels of pgsA-sM2 and pgsA-CTA1-sM2 were monitored by immunoblotting using anti-pgsA, anti-M2 or anti-CT polyclonal antibodies data not shown . To determine the cellular localization of the sM2 and CTA1 proteins expressed on the surface of L. casei via the cell wall anchor protein pgsA, membrane and cytoplasmic fractions were subjected to western blot analysis. As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 .", "As expected, both pgsA-sM2 and pgsA-CTA1-sM2 fusion proteins were detected by anti-pgsA, anti-M2 or anti-CT polyclonal antibodies in the membrane, not in cytoplasmic fractions Fig. 1B, lane 2, 3 and 4 . Immunoreactions were performed with anti-pgsA, and bands representing the size of the fused proteins pgsA-sM2 and pgsA-CTA1-sM2 were detected, while during the reactions with anti-M2 or anti-CT antibodies, no other bands were detected Fig. 1B, lane 3 and 4 . This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei.", "This finding may have resulted from the degradation that occurs during the membrane fractionation procedure. Fluorescence-activated cell sorting FACS and immunofluorescence labeling of the cells were used to verify the localization of the fusion pgsA-sM2 and pgsA-CTA1-sM2 protein on the surface of L. casei. Flow cytometric analysis using rabbit anti-M2 and anti-CT antibodies revealed increase level of fluorescence intensity of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells, compared to that of control L. casei cells Fig. 1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells.", "1C . Immunofluorescence microscopy also showed recombinant bacteria harboring pgsA-sM2 or pgsA-CTA1-sM2 that immunostained positive for sM2 and CTA1, but this was not found in control cells. These results demonstrated that recombinant L. casei could efficiently display the sM2 and CTA1-sM2 fusion proteins on the surface, using pgsA as a membrane anchor protein. Immune Responses Induced by Mucosal Immunization with L. casei Surface Displayed sM2 and CTA1-sM2 Preliminary experiment was conducted to determine the doses and schedule of pgsA-CTA1-sM2/L. casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L.", "casei vaccine candidate on influenza virus protection data not shown . To characterize the immunogenicity of the L. casei surface-displayed sM2 and CTA1conjugated sM2, BALB/c mice were immunized nasally 10 9 cells/20 ml dose or orally 10 10 cells/100 ml dose with recombinant live pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei bacteria. As a negative control, mice were immunized with L. casei harboring the parental plasmid pKV-pgsA pgsA/L. casei and PBS. Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n.", "Serum samples were collected at 0, 14 and 28 days and analyzed by ELISA, using sM2 and CTA1 proteins purified from E. coli as a coating antigen. After the first series of immunization, comparatively low levels of serum IgG were detected both in the i.n. and orally immunized group. However, high antibody levels were detected shortly after the second series of immunization, and the CTA1-conjugated sM2 group induced serum IgG at significant level, compared to sM2-only group and negative controls Fig. 2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig.", "2A and B . Although the conjugation of CTA1 with sM2 was expected to have an adjuvant function only, a significant level of anti-CTA1 antibodies was detected in both the nasal and oral vaccinations Fig. 2A and B right panel . In comparison with the oral group, the nasally immunized group showed higher levels of serum IgG specific to both sM2 and CTA1. To assess the mucosal immune responses, the local IgA levels were determined by ELISA. Lung and intestinal tissues were collected at day 28 of immunization and examined using sM2 protein as a coating antigen. In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L.", "In both routes of vaccination, pgsA-CTA1-sM2/L. casei induced significantly increased levels of sM2specific mucosal IgA compared to the pgsA-sM2/L. casei and control groups. However, as expected, higher levels of antibody titers were detected at the site of inoculation than at the remote site. A similar pattern of antibody responses was observed for both routes of immunization, in which the pgsA-CTA1-sM2/L. casei groups dominated Fig. 2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS.", "2C and D . These data demonstrated that cholera toxin subunit A1-conjugated sM2 resulted in significant enhancements to the sM2-specific IgG and mucosal IgA levels compared with sM2 alone or with controls immunized with pgsA/ L. casei or PBS. Mucosal Immunization with L. casei Surface-displayed sM2 and CTA1-sM2 Stimulated M2-specific Cellular Immune Response To determine whether mucosal vaccination with L. casei surfacedisplayed sM2 and CTA1-conjugated sM2 could induce cellular immunity, IFN-c and IL-4 ELISPOT were performed. Splenocytes from vaccinated mice were stimulated with 10 mg/ml of recombinant sM2 protein or M2 peptide, and the cytokine ELISPOTs were developed. The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n.", "The spots were counted to measure the differences in the CTL responses between the groups. Cells from the mice immunized i.n. with pgsA-CTA1-sM2/L. casei showed significant levels of IFN-c in response to stimulation with sM2 protein and M2 peptide Fig. 3A . Similarly, we observed that i.n. administered groups both for pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei showed detectable levels of IL-4 secreting splenocytes following stimulation with either sM2 protein or M2 peptide Fig. 3B . IFN-c and IL-4 secreting cells were also observed in mice immunized orally with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L.", "casei and pgsA-CTA1-sM2/L. casei Fig. 3C although their levels were lower than i.n. group and were not significant. Control group immunized with pgsA/L. casei showed background spot level for both in intranasal and oral groups. These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability .", "These findings indicate that highly conserved sM2 can induce M2-specific IFN-c and IL-4 secreting T cell responses, while mucosal delivery through L. casei and CTA1 conjugation with sM2 enhanced the cell mediated immunity, which may contribute to broadening the protective immunity. M2 is known as a potential target for the development of broad spectrum influenza vaccine with minimum variability . To confirm the variability of sM2 sequences of the challenged viruses used in this study, we compared the sM2 of influenza subtypes available from U.S. National Center for Biotechnology Information NCBI with our consensus sM2 sequence particularly the whole conserved ecto and some portion of cytoplasmic domain CD although entire CD was included in vaccine construct Table 1 . We found that, viruses used in this study contain 0-8 mismatched amino acids among the amino acids of sM2 compared in this study. To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence.", "To evaluate the efficacy of the sM2 vaccine, week after the final immunization, mice were challenged i.n. with the 10 MLD 50 of A/Aquatic bird/Korea/W81/2005 H5N2 influenza virus subtypes that was homologous to the consensus sM2 sequence. Mice immunized orally with pgsA-sM2/ L. casei and pgsA-CTA1-sM2/L. casei showed 40 and 60% protection respectively. Similarly, i.n. immunization groups conferred 40 and 80%, against the lethal infection with highly virulent H5N2 virus. In contrast, none of the unimmunized mice survived after lethal infection Fig. 4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L.", "4A and B, right panel . Morbidity was increased in the mice immunized via oral route, whereas mice that received i.n. immunization with pgsA-CTA1-sM2/L. casei lost ,20% of their initial body weight and started recovering by 9 day post infection dpi and had completely recovered by day 13 Fig. 4A and B, left panel . We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus.", "We next evaluated the protection efficiency of sM2 vaccine candidate against A/Puerto Rico/8/34 H1N1 , which contains 8 mismatched amino acids relative to the sM2 consensus sequence. Sets of vaccinated mice were challenged with 10 MLD 50 of the H1N1 virus. As shown in figure 4C and D, mice immunized by the The mice were grouped as mentioned in materials and methods and received oral or nasal administrations, according to the schedule. Arrows indicated the immunization routes and periods of pgsA/L. casei, pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization.", "casei or pgsA-CTA1-sM2/L. casei cells. Sera were collected at days 0, 14 and 28; samples from the lungs and intestines were collected at day 28 after immunization. A week after the final immunization, spleens were excised from 3 mice in each group, with one set for CTL analysis. Two or 24 weeks after the last immunization, all mice were challenged with a lethal dose of influenza subtypes through intranasal route and monitored for 13 days. On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry.", "On days 3 and 5 post infection, the lungs were excised from 3 mice in each group to determine the virus titer. On 5 dpi, the mice from one set were sacrificed for lung histopathology and immunohistochemistry. .1371/journal.pone.0094051.g001 CTA1-sM2 Induces Protective Immunity to Pathogenic Influenza A Viruses PLOS ONE | i.n route exhibited a higher level of protection than the orally immunized groups, and mice immunized with pgsA-CTA1-sM2/ L. casei showed a significantly higher level of protection compared to mice immunized with pgsA-sM2/L. casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L.", "casei Fig. 4C and D, right panel . Unimmunized mice lost up to 40% of their body weight and died by 9 dpi. Mice immunized with pgsA-CTA1-sM2/L. casei lost approximately 10% of their body weight, whereas mice immunized with pgsA-sM2/L. casei lost .20% of their initial body weight by 9 dpi and recovered more slowly than mice immunized with pgsA-CTA1-sM2/L. casei Fig. 4C and D, left panel . Another set of vaccinated mice were infected with A/Chicken/ Korea/116/2004 H9N2 to check the range of protection ability of sM2 vaccine induced immune responses. The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L.", "The sM2 sequence of H9N2 contains 2 mismatched relative to the sM2 consensus sequence. The mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight losses and gradual recovery compared to those of mice immunized with pgsA-sM2/L. casei and the unimmunized mice for both the i.n and oral routes Fig. 4E and F left panel . None of the unimmunized mice survived, whereas 100% and 80% of the mice immunized with pgsA-CTA1-sM2/L. casei via the i.n. and oral routes survived, respectively. The survival rates of mice immunized with pgsA-sM2/L. casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel .", "casei were 80% and 60% for the i.n. and oral routes, respectively Fig. 4E and F, right panel . The breadth of protection of the sM2 vaccine against divergent influenza subtypes was also evaluated. Set of immunized mice were challenged with high pathogenic avian influenza HPAI A/ EM/Korea/W149/06 H5N1 , which contains 2 amino acid mismatches relative to the sM2 consensus sequence. Mice immunized via the i.n. and oral routes with pgsA-CTA1-sM2/L. casei showed higher protection efficacies, 80% and 60%, respectively, compared with mice immunized with pgsA-sM2/L. casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel .", "casei, for which the rates were 60% and 20%, respectively Fig. 4G and H, right panel . Regarding morbidity, mice immunized with pgsA-CTA1-sM2/L. casei showed lower morbidity than mice immunized with pgsA-sM2/L. casei Fig. 4G and H, left panel . One more set of vaccinated mice were challenged with the A/Aquatic bird/ Korea/W44/2005 H7N3 virus, which contains 1 mismatch relative to the consensus sM2 sequence, and the body weight and survival were observed for 13 dpi. As shown in figure 4I and J, unimmunized mice lost as much as 30% of their body weight than mice immunized with pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei Fig.", "casei and pgsA-CTA1-sM2/L. casei Fig. 4I and J, left panel . Mice immunized with pgsA-CTA1-sM2/L. casei through the i.n route showed significantly higher level of protection against the H7N3 influenza virus than the other groups Fig. 4I and J, right panel . Taken together, the results indicate that i.n. immunization with pgsA-CTA1-sM2/L. casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi.", "casei induced immune responses that conferred significant levels of protection against divergent subtypes of influenza viruses containing mismatched amino acids ranging from 0 to 8 of the consensus sM2, regardless of whether it was complete or partial. Virus titers in the lungs of challenged mice were measured to estimate replication at 3 and 5 dpi. Mice were immunized via the i.n and oral routes with pgsA-sM2/L. casei and pgsA-CTA1-sM2/ L. casei and challenged with the H5N2, H1N1, H9N2, H5N1 or H7N3 influenza subtypes. On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L.", "On 3 and 5 dpi, 3 mice were sacrificed randomly from each group, and their lung virus titers were measured using the TCID 50 method. Mice immunized with pgsA-CTA1-sM2/L. casei had lower titers at 3 dpi and had significantly reduced viral replication at 5 dpi compared to mice immunized with pgsA-sM2/L. casei or the control groups at the same time Fig. 5A-J . Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 .", "Reduced viral titers in the lungs were observed in groups of mice immunized via the i.n route relative to the mice immunized via the oral route, particularly at day 3 post infections Fig. 5 . These reduced titers may be due to routes of vaccination and challenge being the same, and the titers correlated with the survival results for lethal infections with H5N2, H1N1, H9N2, H5N1 and H7N3. Taken together, these results demonstrate that the consensus sM2 protein fused with CTA1 afforded better protection than sM2, and the i.n route was more potent than the oral route of immunization with regard to protection against a lethal challenge of divergent influenza subtypes. Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope.", "Histopathology and immunohistochemistry were performed to corroborate the lung virus titer findings. At 5 dpi, lungs were randomly collected from each group of one set, fixed and stained with eosin before being examined under a light microscope. As shown in figure 5K , clear signs of profound pulmonary inflammation were observed in the lungs of mice treated with PBS or pgsA/L. casei for both the oral and i.n routes of administration, whereas the lungs of the mice immunized with pgsA-CTA1-sM2/L. casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel .", "casei showed no remarkable pulmonary inflammation compare to the pgsA-sM2/L. casei-treated mice Fig. 5K, middle and left panel . For immunohistochemistry, immunoperoxidase method with an antibody directed against the matrix protein-2 of influenza A virus was used for the detection of virus infected cells in the respective tissues. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. As shown in figure 5K, at 5 days p.i., numerous virusinfected cells were detected in control or pgsA-sM2/L. casei vaccinated mice, whereas highly reduced number of antigen positive cells were found in the mice vaccinated with pgsA-CTA1-sM2/L. casei, both in i.n. and orally immunized group Fig. 5K right panel .", "casei, both in i.n. and orally immunized group Fig. 5K right panel . These results indicate that mice immunized with pgsA-CTA1-sM2/L. casei developed immune responses that are strong enough to inhibit virus replication, which promotes the survival of mice after a lethal infection by influenza A. The PgsA-CTA1-sM2/L. casei Vaccination Induced Longlasting Cross Protection The duration of protection is an important criterion for a potential vaccine. Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig.", "Thus, the longevity of the immunity induced by sM2 and CTA1-conjugated sM2 were investigated by detecting serum IgG and mucosal IgA by ELISA. Significantly increase levels of sM2-specific serum IgG as well as lung and intestinal IgA were observed 180 days after vaccination Fig. 6A and C compare to PBS and pgsA/L. casei groups. Mice were challenged with A/ Aquatic bird/Korea/W81/2005 H5N2 , and the body weight changes and survival were monitored until 13 dpi. The unimmunized mice showed .30% body weight loss Fig. 6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L.", "6B and D left panel and died by day 9 post infection in both the oral and i.n. groups. In contrast, the mice immunized with pgsA-CTA1-sM2/L. casei showed negligible body weight loss, which was recovered by 13 dpi; 80% survived in the i.n. immunized group Fig. 6B right panel , and 60% survived in the orally immunized group Fig. 6D right panel . This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface.", "This result indicates that the CTA1conjugated sM2 mucosal vaccine conferred protection against a lethal infection 6 months after the final immunization. The mucosal immune system is the first immunological barrier against the pathogens that invade the body via the mucosal surface. Thus, the induction of mucosal immunity is necessary to ensure protection against multiple subtypes of influenza A virus. A respiratory virus, influenza A is responsible for annual seasonal epidemics worldwide and, occasionally, pandemics, which are caused by emerging novel subtypes/strains derived through reassortment with avian or porcine viruses. Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine.", "Current influenza vaccines provide strain-specific protection only. Thus, it is crucial to establish a broadly cross-protective influenza vaccine. Antigens that are well conserved among influenza A viruses are considered promising targets for the induction of cross-protection against these different subtypes. However, the goal should be the development of a first line of defense by effectively eliminating pathogens at the mucosal surface. Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain .", "Influenza matrix protein-2 M2 is relatively well conserved among the influenza subtypes and can be considered a promising influenza vaccine antigen . It consists of the following three structural domains: a 24-amino-acid extracellular domain, a 19-amino-acid transmembrane domain, and a 54-amino-acid cytoplasmic tail domain . The extracellular and cytoplasmic domains, which are well conserved among influenza viruses and play an important role in viral assembly and morphogenesis, were used in this study. Here, we developed sM2 consensus derived from the analysis of sequences of H5N1, H1N1 and H9N2 subtypes in the database. Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 .", "Considering the previous findings that extracellular domain particularly aa, 1-13 is highly conserved among the influenza virus subtypes and recognized as epitope for the induction of monoclonal antibodies, which could protect influenza virus infection , sM2 backbone sequence from the H5N1 virus were used. For the possible homology among other subtypes we changed at the position of 14 E-G and 18 R-K and kept unchanged the conserved epitope aa, 1-13 . As shown in sequence alignment, sM2 of consensus sequence has 0-8 mismatches among the subtypes used in this study Table 1 . Moreover, the incorporation of an adjuvant is considered essential to boost the interaction of the vaccine with the mucosal immune system . Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity.", "Various adjuvants, such as liposomes, nanoparticles, and immunostimulating complexes ISCOMs , have been studied and were found to improve the immune response , but their efficacies were not optimal. Despite its potential as a mucosal adjuvant , the use of cholera toxin CT in vaccines is limited by its innate toxicity. Thus, the toxicity of CT would have to be separated from its adjuvanticity before it could be used as a vaccine adjuvant. Studies have shown that constructs consisting of M2e fused with cholera toxin subunit A1 along with a strong ADPribosylating agent and a dimer of the D-fragment of Staphylococcus aureus protein A vaccine elicited complete protection and reduced morbidity . CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus.", "CTA1 retains the adjuvant function of CT without its toxic side effects, such as reactogenicity at the site of its administration and binding to or accumulation in the nervous tissues . Based on previous findings, it has been hypothesized that the consensus sM2 fragment, when fused with the potent mucosal adjuvant CTA1, may induce broad protective immunity against divergent subtypes of influenza virus. In this study, we used the whole 22-kDa CTA1 protein an ADP ribosyltransferase , which consists of three distinct subdomains: CTA11 residues 1 to 132 , CTA12 residues 133 to 161 , and CTA13 residues 162 to 192 . It has been reported that CTA1 lacking CTB has strong adjuvant activities without any toxicity. CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L.", "CTA1 enhances the IgA and IgG antibody responses, as well as CTL activity . For the development of a universal mucosal influenza vaccine with a conserved sM2 peptide and potent adjuvant CTA1, recombinant L. casei displaying sM2 fused with or without CTA1 The lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei showed clear alveoli without inflammatory cell infiltration, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control mice, both of which revealed features of severe pneumonitis middle and left panel . Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L.", "Reduced number of viral antigen were detected in lungs of the mice vaccinated with pgsA-CTA1-sM2/L. casei, in contrast to the lungs of mice vaccinated with pgsA-sM2/L. casei or control revealed features of severe pneumonitis with increase virus antigen right panel . Micrographs are representative for each treatment group at a magnification of 200X. Virus antigen in epithelial cells appears as brown coloration of the nucleus and cytoplasm. In lung titers, bars denote mean 6 S.D. The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB .", "The asterisk indicates a significant difference between pgsA-CTA1-sM2/L. casei and other groups *P,0.05 . .1371/journal.pone.0094051.g005 were constructed for mucosal delivery by the widely used live vaccine vehicle LAB . The pgsA gene used in this study is an anchor for display on the surface of LAB which is derived from the pgsBCA enzyme complex of Bacillus subtilis and consists of transmembrane domain near its N-terminus with the domain located on the outside of the cell membrane. Thus, pgsA is able to cross the cell wall and display the heterologous protein fused to its C-terminus . The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L.", "The developed vaccines were tested through two major routes. We found that vaccination with pgsA-CTA1-sM2/L. casei was able to induce a significantly higher level of sM2-specific serum IgG Fig. 2A and B and mucosal IgA Fig. 2C and D compared to pgsA-sM2/L. casei, and conferring protection against divergent influenza subtypes of both phylogenetic group 1 H1, H5, H9 and group 2 H7 Fig. 4 . This study also revealed that i.n. administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon.", "administration was superior to the oral route of vaccination, which is consistent with other observations . There may be two possible reasons to explain this phenomenon. First, the challenge route is the same as that of the vaccination; specific mucosal IgA can prevent viral colonization in the respiratory tract. Second, the volume of the inocula was 5 times lower than that for oral inoculation, which may have allowed the concentrated form of the antigen to be presented to immune cells. Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments.", "Because greater levels of serum IgG and mucosal IgA were detected in intranasally immunized mice than in those immunized orally Fig. 2 , an alternative explanation could be that the antigens are processed and/or presented differently to immune cells in the two mucosal compartments. Importantly, our study demonstrated for the first time that mucosal immunization with the LAB surface-displayed CTA1-conjugated sM2-based vaccine candidate induced broad protection against challenge with divergent influenza subtypes. However, the mechanism by which Abs against sM2 mediated this broad protection is not fully understood. Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments.", "Previous studies have demonstrated that Abs to the N-terminus of M2e, particularly positions 1-10, inhibited the replication of the influenza A virus . Other studies revealed that anti-M2e IgG-mediated cellular cytotoxicity or phagocytosis can induce the removal of infected cells before progeny virus budding and spread which is supporting our findings of lung virus titer and immunohistochemistry data detected at 5 dpi in our challenge experiments. Therefore, in this study, combination of those responses and Abs to the N-terminus of the sM2 sequence which is conserved among the challenge viruses Table 1 may protect the divergent influenza subtypes after mucosal immunization with the recombinant LAB CTA1-conjugated sM2-based vaccine candidate. Moreover, the cellular immune response plays an important role in controlling viral replication. We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L.", "We examined the Th1-type IFN-c and Th2-type IL-4 cytokine responses by the ELISPOT assay. Significantly higher levels of IFN-c were detected in response to stimulation with both the sM2 protein and M2 peptide in mice immunized with pgsA-CTA1-sM2/L. casei compared to the levels in mice in the pgsA-sM2/L. casei and control groups Fig. 3A and C . Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D .", "Similarly, substantially high levels of IL-4 were observed in mice immunized with pgsA-CTA1-sM2/ L. casei upon stimulation with the sM2 protein and M2 peptide Fig. 3B and D . These results further support the findings that the antibodies and cell-mediated cytotoxicity were specific to the M2 antigen and that their anti-viral activities were induced by monomeric M2, three copies of M2 fused with ASP-1 . Together, these results indicate that sM2 adjuvanted with fused CTA1 induced immune responses in mice, which protected them from divergent influenza subtypes. In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding.", "In this regard, our results have significance for the use of CTA1, which has adjuvant function, in vaccine candidates. As clinical protection is not the only parameter by which vaccine performance is assessed, we evaluated the immunogenicity of the recombinant LAB vaccine on the basis of other parameters, such as the reduction of pathological lesions and virus shedding. In this study, low titers of the challenge virus were titrated from the lungs after vaccination with pgsA-CTA1-sM2/L. casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J .", "casei, whereas challenge virus could be detected at higher titers in the mock mice and those vaccinated with pgsA-sM2/L. casei Fig. 5A-J . Reduced gross and histopathological lesions consistent with viral infection are the primary parameters indicative of influenza vaccine efficacy. Here, we demonstrated that vaccination with pgsA-CTA1-sM2/L. casei remarkably limited the severity of the damage by inhibiting viral replication and the accumulation of inflammatory cells and virus antigen in the lung alveolar tissues, relative to the severity in the unimmunized mice and the mice vaccinated with pgsA-sM2/L. casei Fig. 5K .", "casei Fig. 5K . Our study further demonstrated, for the first time, that recombinant L. casei expressing CTA1-sM2 induced long-lasting immunity and conferred protection against lethal infections by influenza, even at 6 months after the final vaccination Fig. 6 , which is important for any successful vaccine. Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes.", "Similar results were observed in previous studies, in which M2 VLP conferred longterm immunity and cross protection and the antibodies in the sera and mucosal sites were long lived . In conclusion, our findings revealed that the mucosal immunization of mice with recombinant L. casei expressing CTA1conjugated sM2 can induce systemic and local, as well as cellmediated, immune responses against divergent influenza virus subtypes. Thus, the recombinant L. casei expressing CTA1conjugated consensus sM2 mucosal vaccine may be a promising vaccine candidate for influenza pandemic preparedness." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "Nod-like receptor family, pyrin domain-containing 3 NLRP3 regulates the secretion of proinflammatory cytokines interleukin 1 beta IL-1β and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus EMCV 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus SARS-CoV activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion.", "SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K + efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length.", "These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus SARS-CoV , a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame ORF 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins . . The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 .", "The 3 one-third of the genome encodes four structural proteins, spike S , envelope E , matrix M and nucleocapsid N , and non-structural proteins, along with a set of accessory proteins 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b Perlman and Dandekar, 2005; Tan et al., 2005 . SARS-CoV is the etiological agent of SARS Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003 . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor TNF -α, interleukin IL -1β, and IL-6, were detected in autopsy tissues from SARS patients . . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood.", ". Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors PRRs to detect pathogen-associated molecular patterns Medzhitov, 2001; Kawai and Akira, 2010 . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 NLRP3 is activated by a wide variety of stimuli, including virus infection . . Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 .", ". Four models describing activation of the NLRP3 inflammasome have been proposed thus far Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010 . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013 . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals . , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 .", ", fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species ROS or mitochondrial DNA from damaged mitochondria Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012 . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling MAVS or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013 ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain ASC and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 .", "This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion Kayagaki et al., 2015; Shi et al., 2015 . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels Tan et al., 2005; Chen and Ichinohe, 2015 . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains Zeng et al., 2004; Lu et al., 2006 , are thought to act as Na + /K + and K + channels, respectively Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011 , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory.", "Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science The University of Tokyo . Bone marrow-derived macrophages BMMs were prepared as described previously . . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 .", "In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium DMEM; Nacalai Tesque . Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum FBS , and L-glutamine 2 mM at 37 • C/5% CO 2 . HEK293FT cells a human embryonic kidney cell line and HeLa cells a human epithelial carcinoma cell line were maintained in DMEM supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . MDCK cells Madin-Darby canine kidney cells and HT-1080 cells a human fibrosarcoma cell line were grown in Eagle's minimal essential medium E-MEM; Nacalai Tesque supplemented with 10% FBS, penicillin 100 units/ml , and streptomycin 100 µg/ml Nacalai Tesque . Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . .", "Influenza A virus strain A/PR8 H1N1 was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs . . The viral titer was quantified in a standard plaque assay using MDCK cells . . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain . were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan .", "were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang National Taiwan University College of Medicine, Taipei, Taiwan . To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors Invitrogen . To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively . . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets.", ". To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells TOYOBO . To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA .", "HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP green fluorescent protein , or pLenti-M2 using polyethylenimine PEI Max. At 24 h post-transfection, the cells were lysed with RIPA buffer 50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate SDS , 150 mM NaCl and 1 mM EDTA . And the lysates were subjected to SDS-polyacrylamide gel electrophoresis PAGE followed by electroblotting onto polyvinylidene difluoride PVDF membranes. The membranes were incubated over night with mouse anti-V5-tag R960-25, Invitrogen , mouse anti-influenza A virus M2 14C2, Abcam , mouse anti-GFP GF200, Nacalai Tesque , or rabbit antitubulin DM1A, Santa Cruz antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG Jackson Immuno Research Laboratories or anti-rabbit IgG Invitrogen . After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 .", "After washing 3 times with washing buffer 0.05% Tween-20/PBS , the membranes were exposed using Chemi-Lumi One Super Nacalai Tesque , and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus GE Healthcare . To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector Invitrogen using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish 10 cm in diameter were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix Invitrogen using Lipofectamine 2000 Invitrogen . The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter Millipore at 72-96 h post-transfection . . The lentiviral titer was then quantified using HT-1080 cells as described previously .", ". The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection MOI of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 .", "Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay ELISA using paired antibodies eBioscience Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi . . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies .", "At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody M2, Sigma followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG H+L Life Technologies . To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon .", "After washing and blocking, the cells were incubated with rabbit anti-HA 561, MBL and mouse anti-NLRP3 Cryo-2; AdipoGen antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG H+L and Alexa Fluor 568-conjugated goat anti-mouse IgG H+L Life Technologies . Fluorescent signals were observed by confocal microscopy A1R + , Nikon . Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 .", "We previously demonstrated that the influenza virus M2 protein a proton-selective ion channel , its H37G mutant which has lost its proton selectivity and enables the transport of other cations such as Na + and K + , and the EMCV 2B protein a Ca 2+ channel stimulates NLRP3 inflammasome-mediated IL-1β secretion Ichinohe et al., 2010; Ito et al., 2012 . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome Nieto- Torres et al., 2015 . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis Figures 1A-C . We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D .", "We next transduced lipopolysaccharide LPS -primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports Ichinohe et al., Figure 1D . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs Figure 1D . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells Figure 1E . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 .", "These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F located in the transmembrane domain from amino acid residues 7-38 prevent ion conductivity Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012 . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain amino acid residues 127-133 that is involved in the formation of a homodimer to generate the ion channel Lu et al., 2006; Chan et al., 2009 . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein . . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A .", ". To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS Chan et al., 2009; Figure 2A . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report Nieto -Torres et al., 2015 , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs Figure 2B . Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a.", "Notably, the 3a-CS mutant completely abrogated IL-1β secretion Figure 2B , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy.", "Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h lentiviruses or 6 h ATP post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns.", "Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports Yu et al., 2004; Yuan et al., 2005 , the 3a protein localized to the Golgi apparatus Figure 3A . In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum ER Figure 3B . By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3.", "By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures Figures 3A,B . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016 . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells Figure 4 . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation.", "Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel . . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion.", "In addition, K + efflux is a well-known activator of the NLRP3 inflammasome Mariathasan et al., 2006; Petrilli et al., 2007 . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result . , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B .", ", we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM Figure 5A . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins Figure 5B . Since mitochondrial ROS are important for NLRP3 inflammasome activation Nakahira et al., 2011; Zhou et al., 2011 , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009 . As reported previously Nakahira et al., 2011; Ito et al., 2012 , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP Figure 6A . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome.", "Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO Figure 6B . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 .", "Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015 . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015 . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome . . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein.", ". Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 H37G , causing loss of proton selectivity, enables transport of other cations i.e., Na + and K + , thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 EV71 , and human rhinovirus a member of the Picornaviridae family triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments Ito et al., 2012; Triantafilou et al., 2013 . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin Negash et al., 2013; Farag et al., 2017 . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . .", "Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex . . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome Nieto-Torres et al., 2015 . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation . . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 .", ". Although alanine substitution at Cys-133, which is required for dimer or tetramer formation . , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 . , the ion channel activity-loss mutant 3a-CS Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133 Chan et al., 2009 completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane.", "Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane Minakshi and Padhan, 2014 and acts as a K + channel . , thereby presumably stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV Castano-Rodriguez et al., 2018 activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]

[ "BACKGROUND: Mycobacterium tuberculosis MTB is a major cause of morbidity and mortality in the world. To combat against this pathogen, immune cells release cytokines including tumor necrosis factor-α TNF-α , which is pivotal in the development of protective granulomas. Our previous results showed that Bacillus Calmette Guerin BCG , a mycobacterium used as a model to investigate the immune response against MTB, stimulates the induction of TNF-α via mitogen-activated protein kinase MAPK in human blood monocytes. Since MAPK phosphatase-1 MKP-1 is known to regulate MAPK activities, we examined whether MKP-1 plays a role in BCG-induced MAPK activation and cytokine expression. RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1.", "RESULTS: Primary human blood monocytes were treated with BCG and assayed for MKP-1 expression. Our results demonstrated that following exposure to BCG, there was an increase in the expression of MKP-1. Additionally, the induction of MKP-1 was regulated by p38 MAPK and extracellular signal-regulated kinase 1 and 2 ERK1/2 . Surprisingly, when MKP-1 expression was blocked by its specific siRNA, there was a significant decrease in the levels of phospho-MAPK p38 MAPK and ERK1/2 and TNF-α inducible by BCG. CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries .", "CONCLUSIONS: Since TNF-α is pivotal in granuloma formation, the results indicated an unexpected positive function of MKP-1 against mycobacterial infection as opposed to its usual phosphatase activity. Text: Tuberculosis TB remains a major cause of morbidity and mortality in the world, especially in the developing countries . The disease is caused by Mycobacterium tuberculosis MTB and approximately one third of the world's population has been infected by this pathogen. In a recent report, World Health Organization WHO estimated that there are 9.2 million new TB cases around the world in 2006 . In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity .", "In response to MTB infection, induction of cytokines by immune cells is an important defense mechanism. The infected macrophages secrete intercellular signaling factors, proinflammatory cytokines, to mediate the inflammatory response leading to the formation of granuloma and induction of T-cell mediated immunity . In order to understand TB pathogenesis, signaling pathways induced by mycobacteria have long been a subject of interest. Mitogen activated protein kinases MAPKs including extracellular signal-regulated kinase 1 and 2 ERK1/2 , p38 MAPK, and c-Jun N-terminal kinase JNK have been implicated as important cellular signaling molecules activated by mycobacteria . Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR .", "Previous reports have shown that p38 MAPK and ERK1/2 are required in the induction of TNF-α expression in human monocytes infected with M. tuberculosis H37Rv . We have further revealed the significant role of MAPKs in the signal transduction events of mycobacterial activation of primary human blood monocytes PBMo leading to cytokine expressions via the interaction with PKR . However, the subsequent events as to how MAPK is regulated and how such regulation affects cytokine production in response to mycobacteria remain to be elucidated. Since MAPKs are activated by phosphorylation, dephosphorylation of MAPKs seems to be an efficient process to inactivate their activities. It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs .", "It can be achieved by specific protein kinase phosphatases which can remove the phosphate group from MAPKs. Examples of these phosphatases include tyrosine phosphatases, serine/threonine phosphatases, and dual-specificity phosphatases DUSPs . Some DUSPs are also known as MAPK phosphatases MKPs . Currently, there are at least 10 MKPs identified, while MKP-1 is the most studied member of the family. The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment .", "The regulatory role of MKP-1 on cytokine induction is best demonstrated by MKP-1 knockout KO macrophages in response to lipopolysaccharide LPS , a cell wall component of Gram-negative bacteria. MKP-1 KO macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α in response to LPS treatment . Consistent with these results, another group further revealed that LPS-treated MKP-1 KO bone marrow-derived macrophages show increased AP-1 DNA-binding activity . Also, they showed that LPS-induced MKP-1 expression is dependent on myeloid differentiation factor 88 MyD88 and TIR domain-containing adaptor inducing IFN-β TRIF , thus demonstrating the role of MKP-1 in signal transduction. Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection.", "Not only LPS, other TLR inducers including CpG, peptidoglycan, poly IC, and Pam 3 Cys can regulate cytokine expressions including TNF-α, IL-10 via MKP-1 activities . In these processes, MKP-1 serves to mitigate the undesirable effects of septic shock and maintain organ functions by restraining the inflammatory responses following bacterial infection. Another example of MKP-1 function is the immune response to Staphylococcus aureus S. aureus , a Gram positive bacteria. There are higher levels of cytokine production including TNF-α, IL-6, and MIP-1α in MKP-1 KO mice infected with S. aureus . Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria.", "Also, the mice would have a rapid development of multiorgan dysfunction as well as faster mortality rate upon challenge with heat-killed S. aureus . Taken together, these results suggest that MKP-1 protects the host from overactivation of the immune system in response to Gram negative or Gram positive bacteria. In the past, it was believed that different MKP/DUSP family members have overlapping functions. However, the emergence of DUSP2 turned the concept up side down . It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation.", "It was shown that DUSP2 behaves differently and is opposite to the function as stated above. In DUSP2 KO cells, they produced less inflammatory mediators, implying that DUSP2 may play a role in mediating instead of limiting inflammation. For instances, when DUSP2 KO macrophages were treated with LPS, there were less TNF, IL-6, nitric oxide, IL-12-producing cells when compared to those of the wild type counterparts . When the DUSP2 KO bone marrow-derived mast cells were first sensitized with immunoglobulin E IgE receptor FcεRI and then stimulated with dinitrophenol-heat stable antigen, they produced lower TNF mRNA levels, diminished IL-6 production, less phosphorylation of ERK1/2, p38 MAPK, and less transcriptional activities by Elk1 and NFAT-AP-1 . These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK.", "These unexpected positive regulations of immune cell functions by DUSP2 have been hypothesized to be due to crosstalks between MAPKs . Stimulation of KO mast cells and macrophages showed increases in phosphorylation of JNK. Moreover, inhibition of JNK by small molecule inhibitors showed increases in phosphorylation of ERK . The authors also showed that there were physical interactions of DUSP2 with ERK2, DUSP2 with JNK2, as well as DUSP2 and p38 MAPK after stimulation of the cells with dinitrophenol-heat stable antigen. Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses.", "Nevertheless, the details of the crosstalks between MAPKs and phosphatases need further investigation. Thus, the MKP family plays a critical role in the regulation of immune responses. Innate immune response protects the host from MTB infection by secretion of cytokines including TNF-α in immune cells. Meanwhile, MAPK is one of the critical proteins in the regulation of immunity and cytokine expression. Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression.", "Since MAPK is regulated by MKP-1 in response to LPS and the activation of MAPK is important in BCGinduced cytokine expression, we hypothesize that MKP-1 plays a critical role in the immune regulation of BCG in human monocytes. We examined the involvement of MKP-1 in BCG-induced MAPK activation and its consequent cytokine expression. Here, we present evidences that MKP-1 plays an unexpected role in the regulation of cytokine induction by BCG through its control of MAPK phosphorylation. It has been reported that many inducers including growth factors, LPS, peptidoglycan, and dexamethasone can stimulate the expression of MKP-1 in human macrophages, microglia, mast cells or fibroblasts . To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours.", "To investigate the role of different TLR inducers in MKP-1 induction process in human blood monocytes, the level of MKP-1 mRNA was measured by quantitative polymerase chain reaction QPCR method. PBMo were isolated from primary human blood mononuclear cells and stimulated with Pam 3 Cys TLR2 agonist , poly IC TLR3 agonist , or LPS TLR4 agonist for 1 and 3 hours. Following exposure to Pam 3 Cys or LPS, there were significant inductions of MKP-1 mRNA levels within 1 hour of treatment Figure 1A . These effects on MKP-1 induction continued for 3 hours post-treatment with Pam 3 Cys Figure 1A . In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression.", "In contrast, poly IC did not induce MKP-1 Figure 1A . The results indicate that different inducers showed differential up-regulation of MKP-1 expression. LPS has been extensively used to demonstrate the role of MKP-1 in immune response both in vivo and in vitro . To establish a foundation for interpretation of subsequent experimental results, LPS was used as a positive control for the induction of MKP-1 expression. To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours.", "To determine the levels of MKP-1 in response to LPS, kinetics of MKP-1 transcription were determined by QPCR. There was a significant induction of MKP-1 mRNA, which peaked as early as 1 hour upon LPS stimulation, and the levels gradually decreased over a course of 6 hours. These results showed that LPS induced MKP-1 expression Figure 1B . Next, to demonstrate the induction of specific phosphatases by BCG, kinetics of MKP-1 expression in PBMo was studied by using QPCR during BCG treatment. Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A .", "Similar to the results produced by LPS, upon the addition of BCG MOI = 1 CFU/cell , there was a significant induction of MKP-1 mRNA within 1 hour of BCG treatment as determined by Taqman probe specific for MKP-1 Figure 2A . The effects lasted for at least 6 hours Figure 2A . To examine whether the changes of protein production were in parallel to that of the mRNA levels, the protein levels of MKP-1 were measured by Western blotting. In response to BCG, PBMo produced the MKP-1 protein as early as 30 minutes after treatment. The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes.", "The protein levels were maintained for 2 hours and dropped to basal levels at 3 hours Figure 2B . The results demonstrated that there was MKP-1 induction in response to BCG activation in human monocytes. It has been shown that inhibition of p38 MAPK either by specific inhibitor or siRNA reduced the expression of MKP-1 in LPS-or peptidoglycan-treated macrophages . To determine the mechanisms involved in the BCGinduced MKP-1 expression, PBMo were pretreated with several inhibitors including PD98059 inhibitor for MAP kinase kinase MEK or ERK1/2 , SB203580 inhibitor for p38 MAPK , SP600125 inhibitor for JNK , and CAPE inhibitor for NF-κB for 1 hour. A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested.", "A range of concentrations of each inhibitor was used to test their optimal concentrations and effects on cell viability and kinase inhibitions. BCG was added afterwards and total RNA was harvested. The results demonstrated that, with the inhibition of ERK1/2 and p38 MAPK activities by their corresponding relatively specific inhibitors, MKP-1 expressions were significantly reduced Figure 3 . In addition, using higher dose of SB203580, we showed that the inhibition is increased further data not shown . On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2.", "On the contrary, pretreatment of the cells with CAPE and SP600125 did not affect the induction of MKP-1 by BCG Figure 3 . These results suggest that BCG-induced MKP-1 expression is dependent on both p38 MAPK and ERK1/2. Throughout the above experiments, the primary goal was to examine the induction of MKP-1 by BCG in human monocytes. Thus, to further examine the role of MKP-1 in BCG-induced signaling, transfection of siRNA into PBMo was used to knockdown the activity of MKP-1. To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method.", "To demonstrate that the MKP-1 siRNA can indeed knockdown the target gene, PBMo were first transfected with control or MKP-1 siRNA and then treated with BCG for 3 hours. Levels of MKP-1 mRNA were measured by RT-PCR method. In Figure 4A , BCG stimulated MKP-1 expression lanes 1 and 2 . In MKP-1 siRNA transfected monocytes, induction of MKP-1 by BCG was significantly decreased lanes 2 and 4 . The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting.", "The results showed that the siRNA does abrogate the levels of MKP-1 mRNA. To further determine whether MKP-1 siRNA affects BCGinduced MKP-1 at protein levels, PBMo were treated as above and MKP-1 proteins were measured by Western blotting. The results showed that BCG could induce MKP-1 proteins as usual for cells transfected with control siRNA Figure 4B , lanes 1-3 . However, the levels of BCGinduced MKP-1 protein expression were reduced in cells transfected with MKP-1 siRNA Figure 4B , lanes 4-6 . Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS.", "Together, the results suggest that MKP-1 siRNA not only reduced the MKP-1 mRNA in BCG treatment but also abrogated the BCG-induced MKP-1 protein. As stated in the literature , MKP-1 KO mice showed increased TNF-α production in response to LPS. On the basis of the above MKP-1 siRNA results, LPS was then used as a control to demonstrate the effects of this MKP-1 siRNA system. cytokine expression induced by LPS in MKP-1 siRNA transfected cells suggest that the siRNA system is effective in knocking down the MKP-1 expression and MKP-1 acts as a negative regulator in LPS-induced TNF-α expression. To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA.", "To investigate the effect of MKP-1 siRNA on BCG-induced cytokine expression, the levels of TNF-α, IL-6 and IL-10 mRNA were measured by QPCR method. PBMo were transfected with either control or MKP-1 siRNA. Following exposure to BCG with control siRNA, there were significant inductions of TNF-α, IL-6 and IL-10 mRNA levels for 3 hours after treatment as previously reported and data not shown . Next, the effects of MKP-1 siRNA were examined on the cytokine expression induced by BCG. Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells.", "Surprisingly, there was a significant abrogation of BCGinduced TNF-α expression by MKP-1 siRNA Figure 4D . With the knockdown of MKP-1, the level of BCG-induced TNF-α was only 60% compared to that of the control cells, while BCG-induced IL-6 and IL-10 were unchanged in MKP-1 siRNA transfected cells. The results revealed that MKP-1 plays a role in the induction of TNF-α expression upon BCG stimulation, which may be different from that of its conventional functions in which MKP-1 acts as a negative regulator in LPS-induced signaling pathways . The unexpected observations in cytokine expression lead to the investigation on the effects of MKP-1 siRNA on BCG-induced MAPK activation. MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo.", "MKP-1 was found to have a preferential substrate binding to p38 MAPK and JNK than ERK1/2 . The phosphorylation status of MAPKs was assessed in control or MKP-1 siRNA transfected PBMo. Western blotting results demonstrated that BCGinduced both p38 MAPK and ERK1/2 phosphorylation in 15 minutes data not shown and peaked at 30 minutes, and then returned to basal levels in cells treated with the control siRNA Figure 5 . Similar to the results of cytokine expression, phosphorylation of both p38 MAPK and ERK1/2 in response to BCG was decreased in monocytes transfected with MKP-1 siRNA instead of the expected increase in phosphorylation Figure 5 . The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection.", "The results suggest that MKP-1 knockdown would result in reduced MAPK phosphorylation by BCG, implying that the reduced level of TNF-α production in BCG stimulated monocytes is due to reduced phosphorylation of MAPKs by MKP-1 siRNA. This report presented evidences that a novel function of MKP-1 is uncovered in cytokine regulation in response to mycobacterial infection. BCG induces MKP-1 as a rapid response Figure 2 . The induction mechanism of MKP-1 by BCG is dependent on both ERK1/2 and p38 MAPK Figure 3 . Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 .", "Using siRNA approach, the functions of MKP-1 can be examined in primary human monocytes. The results showed that the BCG-induced MAPKs activation as well as cytokine expression are downstream of MKP-1 Figures 4D and 5 . Thus, MKP-1 is a critical signaling molecule that is involved in BCG-induced cytokine expression. Previous reports have shown that MKP-1 induced by LPS or peptidoglycan is dependent on p38 MAPK . Accordingly, BCG-induced MKP-1 can be inhibited by both p38 MAPK and ERK1/2 inhibitors. Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration.", "Interestingly, it has been shown that degradation of MKP-1 is reduced after ERK1/2 phosphorylation . It can be hypothesized that BCG-induced MKP-1 proteins can be stabilized by ERK1/2 and the detailed mechanisms involved require more exploration. Also, since the inhibition of MKP-1 expression by both inhibitors for p38 MAPK and ERK1/ 2 was not complete, it is believed that other proteins may be involved in the BCG-induced MKP-1 expression. On the basis of the literature results on LPS effects Figure 6 , the original expectation for this project is that MKP-1 acts as a negative regulator. LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host.", "LPS-stimulated MKP-1 KO peritoneal macrophages showed prolonged phosphorylation of p38 MAPK and JNK as well as increased production of TNF-α . In doing so, LPS-induced MKP-1 could BCG-induced MAPK phosphorylation is decreased by MKP-1 siRNA prevent prolonged TNF-α production as in sepsis which may lead to severe damage to the host. It was expected that BCG induces MKP-1 and its induction would correlate with the dephosphorylation of MAPKs including p38 MAPK. By blocking the MKP-1 using siRNA, it was expected to have increased p38 MAPK phosphorylation and prolonged TNF-α production in response to BCG. Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA.", "Nevertheless, our results shown here are diametrically opposite. One possibility for the unexpected results may be due to non-specific effects of transfection or siRNA. However, this was not the case since there was a prolonged and increased TNF-α expression after the MKP-1 siRNA-transfected monocytes were treated with LPS Figure 4C . There is now a new hypothesis to explain such paradoxical effects of MKP-1 in TNF-α regulation in which the phosphatase plays a role in positive regulation of TNF-α production in response to BCG as in the case of DUSP2 . The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST .", "The structures of MKP-1 and DUSP2 are similar, with which they both contain a MAPK-interacting domain and a phosphatase catalytic site. By contrast, other DUSP may have extra domains, e.g., PEST . Here, we postulate that the function of MKP-1 in BCG-induced signaling is similar to that of the DUSP2/PAC1. Actually, the discovery of DUSP2 has initially created some paradoxical questions. As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts .", "As described, DUSP2 behaves differently from other MKP family members . In DUSP2 KO macrophages treated with LPS, they produced less inflammatory mediators including less TNF, IL-6, nitric oxide, and IL-12-producing cells, when compared to that of the wild type counterparts . Indeed, the results of these published studies on DUSP2 studies are quite similar to that of our reported results here. It is plausible that these unexpected positive regulations of immune cell functions by DUSP2 were due to crosstalks between MAPKs . It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 .", "It was shown that there are interactions between JNK and ERK1/2 pathways . Here, we showed that the sustained activation of JNK blocks ERK activation Figure 6 . In the DUSP2 situation, stimulation of KO mast cells and macrophages shows increased phosphorylation of JNK, and inhibition of JNK by its own specific inhibitor restores phosphorylation of ERK1/2 . In the BCG-MKP-1 situation, there is an early phosphorylation of p38 MAPK and ERK1/2. Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection.", "Therefore, it is possible that JNK may play a role in the crosstalk interaction of MAPK. However, our preliminary data suggest that the level of phosphorylated JNK was not increased in PBMo MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection Figure 6 MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. LPS model was provided according to literature findings Left . In this scenario, LPS activates MKP-1, which in turn dephosphorylates and deactivates phospho-p38 MAPK, resulting in less TNF-α induction. However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression.", "However, the situation in DHP-HSA activation of DUSP2 is more complicated Middle , since the phosphatase activity causes subsequent inhibition of phospho-JNK which leads to the derepression of phospho-p38 MAPK. Consequently, the combined effects of this cascade results in more TNF-α expression. The unexpected antimycobacterial role of MKP-1 Right may be explained by events similar to the DUSP2 effects. In this case Right , there was an inhibition of unknown pathways or kinases downstream of MKP-1, and the unknown factor in turn inhibits MAPKs activation leading to more TNF-α induction. The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown .", "The details and kinase targets are yet to be identified. transfected with MKP-1 siRNA data not shown . Thus, the details of the crosstalk between MAPKs need further investigation. Here, we present a model to summarize the results and to hypothesize the existence of an as yet unidentified intermediary factor or factors in the pathways downstream of MKP-1 effects in the BCG-induced signaling cascade. The unexpected antimycobacterial role of MKP-1 Figure 6 may be explained by events similar to the DUSP2 effects. In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases.", "In this case, BCG induces MKP-1 expression while also activates MAPKs including p38 MAPK and ERK1/2. Downstream of MKP-1, there is an inhibition of unknown pathways or kinases. The unknown factor in turn inhibits MAPKs activation, which ultimately leads to more TNF-α induction Figure 6 . In summary, MKP-1 plays a critical role in the regulation of cytokine expression upon mycobacterial infection. Inhibition of unknown pathways or kinases downstream of MKP-1, which in turn inhibits MAPKs activation, may be used to explain the novel function of MKP-1 in enhancing MAPK activity and consequent TNF-α expression following BCG treatment Figure 6 . Taken together, the role of MAPK crosstalks need further exploration. .", "Taken together, the role of MAPK crosstalks need further exploration. . TNF-α, 30 cycles TM = 56°C , upstream, 5'-GGCTCCAGGCGGTGCTTGTTC-3', downstream, 5'-AGACGGCGATGCGGCTGATG-3'. PCR products were analyzed on a 1% agarose gel with ethidium bromide and visualized under ultraviolet light. In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA .", "In order to check the size of the PCR products, 1 kb Plus DNA Lad-der™ Invitrogen, USA was run along with the PCR products. To perform QPCR, the levels of MKP-1, and TNF-α mRNA as well as the reference gene GAPDH as internal control were assayed by the gene-specific Assays-on-Demand reagent kits Applied Biosystems, USA . All samples were run in duplicates or triplicates and with no template controls on an ABI Prism 7700 Sequence Detector. The analysis method of QPCR was the comparative cycle number to threshold C T method as described in user bulletin no. 2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T .", "2 of the ABI Prism 7700 Sequence Detection System. The number of C T of the targeted genes was normalized to that of GAPDH in each sample ΔC T . The C T value of the treated cells was compared with that of the untreated or mock-treated cells ΔΔCT . The relative gene expression of the targeted genes fold induction was calculated as 2 -ΔΔCT . Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use.", "Total cellular proteins were extracted by lysing cells in lysis buffer containing 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris-HCl pH 7.4 , 1 mM EDTA, 1 mM EGTA pH 8.0 , 1% SDS, 0.2 mg/ml PMSF, 1 μg/ml aprotinin, 1 mM sodium orthovanadate, 2 μg/ml pepstatin, 2 μg/ml leupeptin, and 50 mM sodium fluoride for 5 minutes. The homogenate was then boiled for 10 minutes and stored at -70°C until use. The concentrations of total protein in cell extracts were determined by BCA™ Protein Assay Kit Pierce, IL, USA . Western blot was done as described . Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies.", "Equal amounts of protein were separated by 10% SDS-PAGE, electroblotted onto nitrocellulose membranes Schleicher & Schuell , and followed by probing with specific antibod-ies for Actin, MKP-1 Santa Cruz Biotech., USA , phospho-p38 MAPK, phospho-ERK1/2 Cell Signaling, USA . After three washes, the membranes were incubated with the corresponding secondary antibodies. The bands were detected using the Enhanced Chemiluminescence System Amersham Pharmacia Biotech as per the manufacturer's instructions. Transfection of siRNA into human monocytes was done as described . MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA .", "MKP-1 siRNA included i MKP1-HSS102982, AAACGCUUCGUAUCCUCCUUUGAGG; ii MKP1-HSS102983, UUAUGCCCAAGGCAUCCAG-CAUGUC; and iii MKP1-HSS102984, UGAUG-GAGUCUAUGAAGUCAAUGGC. MKP-1 knockdown in PBMo was conducted by using MKP1-HSS102983 only or a pool of the above three different MKP-1 Stealth™ Select RNAi ratio = 1:1:1, 200 nM, Invitrogen, USA . Stealth™ RNAi Negative Control Duplex 200 nM was used as a control for sequence independent effects for the siRNA transfection. Transfection of monocytes was done by using jetPEI™ DNA transfection reagent Polyplus Transfection, USA according to the manufacturer's instructions. After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test.", "After transfecting the cells for 24 h, the transfectants were treated with different inducers as described above. Statistical analysis was performed by Student's t test. Differences were considered statistically significant when p values were less than 0.05." ]