bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors
Authors: Hart Bryan E. , Asrican Rose , Lim So-Yon , Sixsmith Jaimie D. , Lukose Regy , Souther Sommer J. R. , Rayasam Swati D. G. , Saelens Joseph W. , Chen Ching-ju , Seay Sarah A. , Berney-Meyer Linda , Magtanong Leslie , Vermeul Kim , Pajanirassa Priyadharshini , Jimenez Amanda E. , Ng Tony W. , Tobin David M. , Porcelli Steven A. , Larsen Michelle H. , Schmitz Joern E. , Haynes Barton F. , Jacobs William R. , Lee Sunhee , Frothingham Richard 
Published: April 2015

ABSTRACTThe well-established safety profile of the tuberculosis vaccine strain,Mycobacterium bovisbacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCDtransformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging bothin vitroandin vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCDvaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplificationin vitroand following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplificationin vitroand induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCDlots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.
Antiviral Innate Immune Activation in HIV-Infected Adults Negatively Affects H1/IC31-Induced Vaccine-Specific Memory CD4+T Cells
Authors: Lenz Nicole , Schindler Tobias , Kagina Benjamin M. , Zhang Jitao David , Lukindo Tedson , Mpina Maxmillian , Bang Peter , Kromann Ingrid , Hoff Søren T. , Andersen Peter , Reither Klaus , Churchyard Gavin J. , Certa Ulrich , Daubenberger Claudia A. 
Published: April 2015

ABSTRACTTuberculosis (TB) remains a global health problem, with vaccination being a necessary strategy for disease containment and elimination. A TB vaccine should be safe and immunogenic as well as efficacious in all affected populations, including HIV-infected individuals. We investigated the induction and maintenance of vaccine-induced memory CD4+T cells following vaccination with the subunit vaccine H1/IC31. H1/IC31 was inoculated twice on study days 0 and 56 among HIV-infected adults with CD4+lymphocyte counts of >350 cells/mm3. Whole venous blood stimulation was conducted with the H1 protein, and memory CD4+T cells were analyzed using intracellular cytokine staining and polychromatic flow cytometry. We identified high responders, intermediate responders, and nonresponders based on detection of interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) expressing central (TCM) and effector memory CD4+T cells (TEM) 182 days after the first immunization. Amplicon-based transcript quantification using next-generation sequencing was performed to identify differentially expressed genes that correlated with vaccine-induced immune responses. Genes implicated in resolution of inflammation discriminated the responders from the nonresponders 3 days after the first inoculation. The volunteers with higher expression levels of genes involved in antiviral innate immunity at baseline showed impaired H1-specific TCMand TEMmaintenance 6 months after vaccination. Our study showed that in HIV-infected volunteers, expression levels of genes involved in the antiviral innate immune response affected long-term maintenance of H1/IC31 vaccine-induced cellular immunity. (The clinical trial was registered in the Pan African Clinical Trials Registry [PACTR] with the identifier PACTR201105000289276.)
Evaluation of the Salmonella enterica Serovar Pullorum Pathogenicity Island 2 Mutant as a Candidate Live Attenuated Oral Vaccine
Authors: Yin Junlei , Cheng Zhao , Wang Xiaochun , Xu Lijuan , Li Qiuchun , Geng Shizhong , Jiao Xinan 
Published: April 2015

ABSTRACTSalmonella entericaserovar Pullorum (S. Pullorum) is a highly adapted pathogen that causes pullorum disease (PD), an important systemic disease of poultry that causes severe economic losses in developing countries. In the interests of developing a safe and immunogenic oral vaccine, the efficacy of aSalmonellapathogenicity island 2 (SPI2)-deleted mutant ofS. Pullorum (S06004ΔSPI2) was evaluated in chickens. S06004ΔSPI2 was severely less virulent than the parental wild-type strain S06004 as determined by the 50% lethal dose (LD50) for 3-day-old chickens when injected intramuscularly. Two-day-old chickens immunized with a single oral dose of S06004ΔSPI2 showed no differences in body weight or clinical symptoms compared with those in the negative-control group. S06004ΔSPI2 bacteria were not isolated from livers or spleens of immunized chickens after a short period of time, and specific humoral and cellular immune responses were significantly induced. Immunized chickens were challenged withS. Pullorum strain S06004 andSalmonellaenterica serovar Gallinarum (S. Gallinarum) strain SG9 at 10 days postimmunization (dpi), and efficient protection against the challenges was observed. None of the immunized chickens died, the clinical symptoms were slight and temporary following challenge in immunized chickens compared with those in the control group, and these chickens recovered by 3 to 5 dpi. Overall, these results demonstrate that S06004ΔSPI2 can be used as a live attenuated oral vaccine.
A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model
Authors: Anosova Natalie G. , Cole Leah E. , Li Lu , Zhang Jinrong , Brown Anna M. , Mundle Sophia , Zhang Jianxin , Ray Satyajit , Ma Fuqin , Garrone Pierre , Bertraminelli Nicola , Kleanthous Harry , Anderson Stephen F. 
Published: April 2015

ABSTRACTClostridium difficileinfection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and Bin vitroand because of theirin vivoefficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains ofC. difficiletoxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.
Identification of Haemophilus influenzae Type b Isolates by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry
Authors: Månsson Viktor , Resman Fredrik , Kostrzewa Markus , Nilson Bo , Riesbeck Kristian 
Published: April 2015

ABSTRACT

Haemophilus influenzae
type b (Hib) is, in contrast to non-type b
H. influenzae
, associated with severe invasive disease, such as meningitis and epiglottitis, in small children. To date, accurate
H. influenzae
capsule typing requires PCR, a time-consuming and cumbersome method. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) provides rapid bacterial diagnostics and is increasingly used in clinical microbiology laboratories. Here, MALDI-TOF MS was evaluated as a novel approach to separate Hib from other
H. influenzae
. PCR-verified Hib and non-Hib reference isolates were selected based on genetic and spectral characteristics. Mass spectra of reference isolates were acquired and used to generate different classification algorithms for Hib/non-Hib differentiation using both ClinProTools and the MALDI Biotyper software. A test series of mass spectra from 33 Hib and 77 non-Hib isolates, all characterized by PCR, was used to evaluate the algorithms. Several algorithms yielded good results, but the two best were a ClinProTools model based on 22 separating peaks and subtyping main spectra (MSPs) using MALDI Biotyper. The ClinProTools model had a sensitivity of 100% and a specificity of 99%, and the results were 98% reproducible using a different MALDI-TOF MS instrument. The Biotyper subtyping MSPs had a sensitivity of 97%, a specificity of 100%, and 93% reproducibility. Our results suggest that it is possible to use MALDI-TOF MS to differentiate Hib from other
H. influenzae
. This is a promising method for rapidly identifying Hib in unvaccinated populations and for the screening and surveillance of Hib carriage in vaccinated populations.
Escherichia coli O-Genotyping PCR: a Comprehensive and Practical Platform for Molecular O Serogrouping
Authors: Iguchi Atsushi , Iyoda Sunao , Seto Kazuko , Morita-Ishihara Tomoko , Scheutz Flemming , Ohnishi Makoto 
Published: April 2015

ABSTRACT

The O serogrouping of pathogenic
Escherichia coli
is a standard method for subtyping strains for epidemiological studies and enhancing phylogenetic studies. In particular, the identification of strains of the same O serogroup is essential in outbreak investigations and surveillance. In a previous study, we analyzed the O-antigen biosynthesis gene cluster in all known
E. coli
O serogroups (A. Iguchi et al., DNA Res, 22:101–107, 2015,
http://dx.doi.org/10.1093/dnares/dsu043
). Based on those results, we have arranged 162 PCR primer pairs for the identification or classification of O serogroups. Of these, 147 pairs were used to identify 147 individual O serogroups with unique O-antigen biosynthesis genes, and the other 15 pairs were used to identify 15 groups of strains (Gp1 to Gp15). Each of these groups consisted of strains with identical or very similar O-antigen biosynthesis genes, and the groups represented a total of 35 individual O serogroups. We then used the 162 primer pairs to create 20 multiplex PCR sets. Each set contained six to nine primer pairs that amplify products of markedly different sizes. This genetic methodology (
E. coli
O-genotyping PCR) allowed for comprehensive, rapid, and low-cost typing. Validation of the PCR system using O-serogroup references and wild strains showed that the correct O serogroups were specifically and accurately identified for 100% (182/182) and 90.8% (522/575) of references and wild strains, respectively. The PCR-based system reported here might be a promising tool for the subtyping of
E. coli
strains for epidemiological studies as well as for the surveillance of pathogenic
E. coli
during outbreaks.
SAMHD1 Limits HIV-1 Antigen Presentation by Monocyte-Derived Dendritic Cells
Authors: Ayinde Diana , Bruel Timothée , Cardinaud Sylvain , Porrot Françoise , Prado Julia G. , Moris Arnaud , Schwartz Olivier 
Published: April 2015

ABSTRACT

Monocyte-derived dendritic cells (MDDC) stimulate CD8
+
cytotoxic T lymphocytes (CTL) by presenting endogenous and exogenous viral peptides via major histocompatibility complex class I (MHC-I) molecules. MDDC are poorly susceptible to HIV-1, in part due to the presence of SAMHD1, a cellular enzyme that depletes intracellular deoxynucleoside triphosphates (dNTPs) and degrades viral RNA. Vpx, an HIV-2/SIVsm protein absent from HIV-1, antagonizes SAMHD1 by inducing its degradation. The impact of SAMHD1 on the adaptive cellular immune response remains poorly characterized. Here, we asked whether SAMHD1 modulates MHC-I-restricted HIV-1 antigen presentation. Untreated MDDC or MDDC pretreated with Vpx were exposed to HIV-1, and antigen presentation was examined by monitoring the activation of an HIV-1 Gag-specific CTL clone. SAMHD1 depletion strongly enhanced productive infection of MDDC as well as endogenous HIV-1 antigen presentation. Time-lapse microscopy analysis demonstrated that in the absence of SAMHD1, the CTL rapidly killed infected MDDC. We also report that various transmitted/founder (T/F) HIV-1 strains poorly infected MDDC and, as a consequence, did not stimulate CTL. Vesicular stomatitis virus glycoprotein (VSV-G) pseudotyping of T/F alleviated a block in viral entry and induced antigen presentation only in the absence of SAMHD1. Furthermore, by using another CTL clone that mostly recognizes incoming HIV-1 antigens, we demonstrate that SAMHD1 does not influence exogenous viral antigen presentation. Altogether, our results demonstrate that the antiviral activity of SAMHD1 impacts antigen presentation by DC, highlighting the link that exists between restriction factors and adaptive immune responses.

IMPORTANCE
Upon viral infection, DC may present antigens derived from incoming viral material in the absence of productive infection of DC or from newly synthesized viral proteins. In the case of HIV, productive infection of DC is blocked at an early postentry step. This is due to the presence of SAMHD1, a cellular enzyme that depletes intracellular levels of dNTPs and inhibits viral reverse transcription. We show that the depletion of SAMHD1 in DCs strongly stimulates the presentation of viral antigens derived from newly produced viral proteins, leading to the activation of HIV-1-specific cytotoxic T lymphocytes (CTL). We further show in real time that the enhanced activation of CTL leads to killing of infected DCs. Our results indicate that the antiviral activity of SAMHD1 not only impacts HIV replication but also impacts antigen presentation by DC. They highlight the link that exists between restriction factors and adaptive immune responses.
Recombination of Globally Circulating Varicella-Zoster Virus
Authors: Norberg Peter , Depledge Daniel P. , Kundu Samit , Atkinson Claire , Brown Julianne , Haque Tanzina , Hussaini Yusuf , MacMahon Eithne , Molyneaux Pamela , Papaevangelou Vassiliki , Sengupta Nitu , Koay Evelyn S. C. , Tang Julian W. , Underhill Gillian S. , Grahn Anna , Studahl Marie , Breuer Judith , Bergström Tomas 
Published: April 2015

ABSTRACT

Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine
in vivo
, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either
in vivo
or
in vitro
during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade.

IMPORTANCE
Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.
Differential Expression of CX3CL1 in Hepatitis B Virus-Replicating Hepatoma Cells Can Affect the Migration Activity of CX3CR1
+
Immune Cells
Authors: Kondo Yasuteru , Kimura Osamu , Tanaka Yasuhito , Ninomiya Masashi , Iwata Tomoaki , Kogure Takayuki , Inoue Jun , Sugiyama Masaya , Morosawa Tatsuki , Fujisaka Yasuyuki , Shimosegawa Tooru 
Published: April 2015

ABSTRACT

In addition to stellate cells and immune cells, inflamed hepatocytes and hepatoma cells express various kinds of chemokines that attract various kinds of immune cells. Previously, we reported that hepatitis B virus (HBV) replication can induce physiological stress. The aim of this study was to analyze the effect of chemokines produced by HBV-infected hepatocytes and hepatoma cells. A real-time PCR array targeting genes related to chemokines and enzyme-linked immunosorbent assay (ELISA) were carried out to detect the specific chemokines produced by Huh7 cells and HepG2 cells infected with various HBV genotypes. A migration assay, flow cytometry analysis, and immunohistochemistry were carried out to analyze the candidate immune cells that can affect the immunopathogenesis of HBV infection. The expressions of CX3CL1 mRNA and protein were significantly different among HBV genotypes A, B, and C and control cells (mock) (
P
< 0.05). CD56
+
NK cells and CD8
+
T cells migrated to the hepatoma cells with HBV replication. Moreover, the migration activity of both immune cells was partially cancelled after the treatment of CX3CL1 neutralizing antibody. The expression level of NKG2D on CX3CR1
+
NK cells in HCC with HBV infection was significantly lower than that in hepatocellular carcinoma (HCC) with HCV infection and chronic hepatitis B and C patients (
P
< 0.05). On the other hand, the frequency of PD-1
high
CX3CR1
+
CD8
+
T cells in HCC with HBV infection was significantly higher than that in HCC with HCV infection and chronic hepatitis B and C (
P
< 0.05). The expression of CX3CL1 in HBV-replicating hepatocytes and hepatoma cells could contribute to the immunopathogenesis of HBV infection.

IMPORTANCE
The progressions of the disease are significantly different among HBV genotypes. However, it has not been clear that how different HBV genotypes could induce different inflammatory responses. Here, we first report that the levels of expression of CX3CL1 mRNA and protein were significantly different among HBV genotypes A, B, and C and mock. Not only the differential expression of CX3CL1 among the genotypes but also the phenotype of CX3CR1
+
NK cells and T cells were gradually changed during the progression of the disease status. In addition to
in vitro
study, the analysis of immunohistochemistry with human samples and NOG mice with human lymphocytes and hepatoma cells supports this phenomenon. The quantification of CX3CL1 could contribute to better understanding of the disease status of HBV infection. Moreover, modifying CX3CL1 might induce an immune response appropriate to the disease status of HBV infection.
Human Plasmacytoid Dendritic Cells Elicited Different Responses after Infection with Pathogenic and Nonpathogenic Junin Virus Strains
Authors: Negrotto Soledad , Mena Hebe A. , Ure Agustin E. , Jaquenod De Giusti Carolina , Bollati-Fogolín Mariela , Vermeulen Elba M. , Schattner Mirta , Gómez Ricardo M. 
Published: April 2015

ABSTRACT
The arenavirus Junin virus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. We characterized the JUNV infection of human peripheral blood-derived plasmacytoid dendritic cells (hpDC), demonstrating that hpDC are susceptible to infection with the C#1 strain (attenuated) and even more susceptible to infection with the P (virulent) JUNV strain. However, hpDC elicited different responses in terms of viability, activation, maturation, and cytokine expression after infection with both JUNV strains.
A Seminomadic Population in Bangladesh with Extensive Exposure to Macaques Does Not Exhibit High Levels of Zoonotic Simian Foamy Virus Infection
Authors: Craig Karen L. , Hasan M. Kamrul , Jackson Dana L. , Engel Gregory A. , Soliven Khanh , Feeroz Mostafa M. , Wang Xiaoxing , Jones-Engel Lisa , Linial Maxine L. 
Published: April 2015

ABSTRACT
Simian foamy viruses (SVF) are ubiquitous in nonhuman primates (NHP). SFV can be zoonotically transmitted to humans who either work with or live commensally with NHP. We analyzed the blood of 45 Bangladeshi performing monkey owners (an ethnic group called the Bedey) for SFV infection. Surprisingly, a PCR assay failed to detect SFV infection in any of these participants. This is in contrast to our previously reported infection rate of about 5% among Bangladeshi villagers.