Patent ID: 12214030

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the use of an outer membrane vehicle as a new plague vaccine. More specifically, a vaccine platform according to the present invention was developed and tested using aYersinia pestismutant synthesizing an adjuvant form lipid A (monophosphoryl lipid A, MPLA) that largely increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, an Asd-based balanced-lethal host-vector system was constructed to oversynthesize the LcrV antigen ofY. pestis, raise the amounts of LcrV enclosed in OMVs by Type II secretion system, and eliminate harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. As described herein, vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge and intranasal challenge and was significantly superior to that resulting from vaccination with LcrV/alhydrogel. Self-adjuvantingY. pestisOMVs are therefore a new plague vaccine candidate and that the design of OMVs according to the present invention could serve as a robust approach for vaccine development. For instance, OMVs may be used to induce an immune response to one or more of the pathogensY. pestis, Y. pseudotuberculosis, andY. enterocolitica. Advantageously, OMVs can deliver heterologous antigens of other pathogens (such asB. anthracis) and may be used to prevent corresponding diseases in animals and humans.

The present invention comprises certain recombinantY. pestisstrains. Typically, the bacterium is derived fromY. pestisKIM6+. Alternatively, a bacterium of the invention may be a strain listed in Table 1.

SeveralYersiniaspecies are amenable for use in the present invention. In one embodiment, a recombinantYersiniabacterium of the invention may be aY. pestisbacterium. In another embodiment, a recombinantYersiniabacterium of the invention may be aY. pseudotuberculosisorY. enterocoliticabacterium. The Δasd, ΔyrbE, ΔtolB, Δlpp, ΔnlpI, ΔlacZ::caf1R-caf1M-caf1A-caf1 may be introduced intoY. pseudotuberculosisorY. enterocoliticato achieve hyper-vesiculation in bacteria and produce high amounts of OMVs. In addition, the Δyops (cure of pYV plasmid that is similar to pCD1) would be introduced intoY. pseudotuberculosisorY. enterocoliticato eliminate potential immune suppression caused by these virulence factors and enhance protective immune response of OMVs against pathogens. In yet another embodiment, a recombinantYersiniabacterium may be aY. pestisorY. pseudotuberculosisbacterium, such as YPS or YPtbS listed in Table 1 and Table 4.

The present invention encompasses a recombinantYersiniabacterium capable of adapted an Asd+ plasmid using a balance-lethal system to over-synthesize protective antigens and generate OMVs containing these antigens. “OMVs” as used herein, Bacterial outer membrane vesicles (OMVs) are vesicles of lipids released from the outer membranes of bacteria. These vesicles may be involved in trafficking bacterial cell signaling biochemicals, which may include DNA, RNA, proteins, endotoxins and allied virulence molecules. OMVs have multiple mechanisms whereby they interact with and regulate innate immune responses to facilitate the onset of bacterial pathogenesis in the host, also OMVs can modulate adaptive immune responses to bacterial pathogens via multiple mechanisms.

A bacterium capable of vesiculation, Vesiculation is a ubiquitous secretion process of Gram-negative bacteria, where outer membrane vesicles (OMVs) are small spherical particles on the order of 30 to 300 nm composed of outer membrane (OM) and lumenal periplasmic content. In one embodiment of the invention, LpxE, a 1-dephosphase fromFrancisella novicida, is able to remove 1-phosphate of lipid A. The bacterium with the lpxE expression can produce monophosphate lipid A (MPLA) and significantly increase bacterial vesiculation, resulting high OMV production. In a preferred embodiment of the invention, such hyper-vesiculation can be achieved by disrupting certain genes ΔyrbE, ΔtolR, Δlpp, and ΔnlpI, that are associated with maintenance of bacterial membrane integrity.

As used herein, “antigen” refers to a biomolecule capable of eliciting an immune response in a host. In some embodiments, an antigen may be a protein, or fragment of a protein, or a nucleic acid. In an exemplary embodiment, the antigen elicits a protective immune response. As used herein, “protective” means that the immune response contributes to the lessening of any symptoms associated with infection of a host with the pathogen the antigen was derived from or designed to elicit a response against. For example, a protective antigen from a pathogen, such asBacillus anthracis, may induce an immune response that helps to ameliorate symptoms associated withB. anthracisinfection or reduce the morbidity and mortality associated with infection with the pathogen. The use of the term “protective” in this invention does not necessarily require that the host is completely protected from the effects of the pathogen.

Some examples of microorganisms useful as a source for antigen are listed below. These may include microorganisms for the control of plague caused byYersinia pestisand otherYersiniaspecies such asY. pseudotuberculosisandY. enterocolitica, for the control of gonorrhea caused byNeisseria gonorrhoea, for the control of syphilis caused byTreponema pallidum, and for the control of venereal diseases as well as eye infections caused byChlamydia trachomatis. Species ofStreptococcusfrom both group A and group B, such as those species that cause sore throat or heart diseases,Erysipelothrix rhusiopathiae, Neisseria meningitidis, Mycoplasma pneumoniaeand otherMycoplasma-species,Hemophilus influenza, Bordetella pertussis, Mycobacterium tuberculosis, Mycobacterium leprae, otherBordetellaspecies,Bacillus anthracis, Clostridium difficile, Clostridium perfringens, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Streptococcus equi, Streptococcus pneumoniae, Brucella abortus, Pasteurella hemolyticaandP. multocida, Vibrio cholera, Shigella., RNA viruses, for example from the classes, influenza viruses Papovavirus, Adenovirus, Herpesvirus, Poxvirus, Parvovirus, Reovirus, Picornavirus, Myxovirus, Paramyxovirus, Flavivirus or Retrovirus (including HIV). Antigens may also be derived from pathogenic fungi, protozoa, parasites and human cancers.

A suitable antigen derived fromYersiniaand designed to induce an immune response againstYersiniamay include LcrV, F1, Psn and Ail. LcrV ofYersiniais a 37-kDa multifunctional protein that has been shown to act at the level of secretion control by binding the Ysc inner-gate protein LcrG and to modulate the host immune response by altering cytokine production. LcrV also is essential for the unidirectional targeting of Yops to the cytosol of infected eukaryotic cells. A promising subunit vaccine is based on LcrV. Active immunization with purified V antigen or passive immunization with antiserum against V antigen provides protection against plague in mice. CD8+ T-cell immune responses primed to LcrV appear to confer protection againstY. pestisin mice. In one embodiment, a live attenuatedY. pseudotuberculosisused as a vector to inject the LcrV antigen fromY. pestisvia T3SS elicits both antibody responses and specific T-cell responses to LcrV ofY. pestis, resulting in enhanced protective immunity against plague.

In another embodiment,Yersinia pestisuses its F1 capsule to enhance survival and cause virulence to mammalian hosts.Y. pestisexpresses the caf operon (encoding the F1 capsule) in a temperature-dependent manner. Since F1 is produced in large quantities and secreted into the host tissues, it also serves as a major immune target. Immunity to infection has been correlated with the presence of antibody to the capsular F1 antigen, and immunization with the F1 antigen induces protection against the disease in animal models. A live attenuatedY. pseudotuberculosisstrain with the caf operon inserted into its chromosome to synthesize F1 in a temperature-dependent manner, can enhance its immunogenicity.

In another embodiment, Pesticin receptor (Psn), an outer membrane protein that is chromosomally in the high pathogenicity island which is present only in highly pathogenic strains ofYersiniasuch asY. enterocolitica1B,Y. pseudotuberculosisandY. pestis. Psn is part of an inorganic iron transport system. Psn as an antigen can stimulate protective immune response againstY. pestisinfection.

In an exemplary embodiment, a bacterium of the invention may comprise one or more mutations selected from the group comprising Δasd, ΔlacZ::caf1R-caf1M-caf1Δ-caf1, ΔyrbE, ΔtolR, Δlpp, and ΔnlpI.

In another embodiment, a bacterium of the invention harboring a plasmid may comprise multiple antigens fromYersinia, such as LcrV, Psn, YopD, andBacillus anthracis, such as PA, LF, EF, and exosporium antigen BxpB.

A recombinant bacterium of the invention may be administered to a host as a vaccine composition. As used herein, a vaccine composition may be a composition designed to elicit an immune response againstYersinia. Additionally, a vaccine composition may be a composition designed to elicit an immune response againstYersiniaand against one or more additional pathogens, such as,Brucella, B. anthracis, Clostridium, Francisella, Burkholderia, Borrelia, E. coli, Salmonella, Staphylococcus, pseudomonasorKlebsiella. In an exemplary embodiment, the immune response is protective, as described above.

Vaccine compositions of the present invention may be administered to any host capable of mounting an immune response. Such hosts may include all vertebrates, for example, mammals, including domestic animals, agricultural animals, laboratory animals, humans, and rarely in cold-blood animals.

In exemplary embodiments, OMVs from the recombinant bacterium is alive when administered to a host in a vaccine composition of the invention. Suitable vaccine composition formulations and methods of administration are detailed below.

A vaccine composition comprising a recombinant bacterium of the invention may optionally comprise one or more possible additives, such as carriers, preservatives, stabilizers, adjuvants (CpG, polyI:C, c-di-GMP, or Curdlan), and other substances.

The dosages of a vaccine composition of the invention can and will vary depending on the antigen amounts in OMVs, the intended host, and immunization route, as will be appreciated by one of skill in the art. Generally, the dosage need only be sufficient to elicit a protective immune response in a majority of hosts. Routine experimentation may readily establish the required dosage. Typical initial dosages of vaccine for intramuscular injection could be about 50 to 100 μl depending upon the preparation of OMVs. Administering multiple dosages may also be used as needed to provide the desired level of protective immunity.

A vaccine of the invention may be administered via any suitable route, such as by intradermal, intramuscular, subcutaneous or intranasal administration. Additionally, other methods of administering the OMVs, such as, oral administration or other parenteral routes, are possible.

A further aspect of the invention encompasses methods of using an OMV of the invention. For instance, in one embodiment the invention provides a method for modulating a host's immune system. The method comprises administering to the host an effective amount of a composition comprising an OMV of the invention. One of skill in the art will appreciate that an effective amount of a composition is an amount that will generate the desired immune response (e.g., innate, mucosal, humoral or cellular). The monitoring of the response can be by quantitating the titers of antibodies or lymphocytes recognizing the selected antigens or by demonstrating and measuring the level of protective immunity.

In still another embodiment, an OMV of the invention may be used in a method for eliciting an immune response againstYersiniaand one or more additional pathogens in an individual in need thereof. The method comprises administrating to the host an effective amount of a composition comprising an OMV as described herein.

In a further embodiment, an OMV described herein may be used in a method for ameliorating one or more symptoms of bubonic plague, pneumonic plague, yersiniosis, or anthrax in a host in need thereof. The method comprises administering an effective amount of a composition comprising an OMV as described herein.

EXAMPLE 1

Materials and Methods

Bacterial strains, plasmids, culture conditions, and molecular operations. All bacterial strains and plasmids used in this study are listed in Table 1 and Table 2 below. All bacterial cultures and molecular procedures as in the Supplementary Information below.

TABLE 1Strains and plasmids used in this studyStrain or PlasmidGenotype or relevant characteristicsStrainsE. coliX6212F-λ- ϕ80 Δ(lacZYA-argF) endA1 recA1 hsdR17 deoR thi-1glnV44 gyrA96 relA1 ΔasdA4E. coliX7213thi-1 thr-1 leuB6 fhuA21 lacY1 glnV44 ΔasdA4 recA1 RP4 2-Tc::Mu [λpir]; KmrY. pestisKIM6+ (pCD1Ap)pCD1Ap, pMT1, pPCP1, Pgm+KIM6+pCD1−pMT1, pPCP1, Pgm+χ10015ΔlpxP:: PlpxLlpxLχ10027ΔlpxP:: PlpxLlpxL ΔlacZ:: PlpplpxEYPS1Δasd12 KIM6+YPS2Δasd12χ10015YPS3Δasd12χ10027YPS4Δasd12 Δymt50 KIM6+YPS5Δasd12 Δ ymt50χ10015YPS6Δasd12Δ ymt50χ10027YPS7Δasd12 Δymt50 KIM6+ pPCP1−YPS8Δasd12 Δymt50χ10015 pPCP1−YPS9Δasd12 Δymt50χ10027 pPCP1−PlasmidspRE112Suicide vector, Cmr, mob−(RP4)R6K ori, sacBpYA3342Asd+; pBR oripYA3493Asd+; β-lactamase signal sequence-based periplasmicsecretion, pBR oripYA4373The cat-sacB cassette in sites of PstI and SacI pUC18pSMV12The full-lengthY. pestislcrV was cloned into pYA3342pSMV13The full-lengthY. pestislcrV was cloned into pYA3620pSMV25The flanking regions of Δasd ofY pestisinto XmaI and KpnIsites of pRE112pSMV26The replication origin of pPCP1 cloned into pYA4373

TABLE 2Primers used in this workNameSequencelcrV-1cgggaattcatgattagagcctacgaaca(EcoRI)(SEQ ID NO: 1)lcrV-2atgattagagcctacgaaca(SEQ ID NO: 2)lcrV-3cggaagctttcatttaccagacgtgtcatctag(HindIII)(SEQ ID NO: 3)Asd-1cggggtaccggaaatgggcgatgccgtagtcgcg(KpnI)(SEQ ID NO: 4)Asd-2acgctatgcgccgctaaaaaatagtgtttactgccctgccttggaagg (SEQ ID NO: 5)Asd-3cagggcagtaaacactattttttagcggcgcatagcgtgtcatatcgt (SEQ ID NO: 6)Asd-4cggcccgggtcgaggagaccgaccagagcctcg(XmaI) (SEQ ID NO: 7)pPCP1-Fattaggatccatcactgacggagcacaacgg(EcoRI) (SEQ ID NO: 8)pPCP1-Rgccgaagctttgttaccgcagcaatacccat(HindIII) (SEQ ID NO: 9)

OMV isolation. OMVs were isolated fromY. pestisstrains as previously described with minor modifications. Briefly, the strains were grown at 28° C. in heart Infusion broth (Difco) for 14 h and then incubated at 37° C. for 4 h. The bacterial cultures were supplemented with EDTA (pH 8.0) at 100 mM and kept on ice for 1 h. Then, the bacterial cells were pelleted by centrifugation at 10,000×g at 4° C. for 20 min. The culture supernatant was filtered using a 0.45 μm pore membrane (Millipore) to remove the residual bacterial cells and concentrated with a 100 kDa filter using a Vivaflow 200 system (Sartorius). The OMVs were harvested by ultracentrifugation (120,000×g) for 2 h at 4° C. The vesicle pellet was washed and resuspended in 0.1× sterilized PBS (pH 7.4), and the ultracentrifugation step was repeated. The final vesicle pellet was resuspended in 0.1× sterilized PBS, filtered with a 0.22 μm pore membrane (Millipore) and stored at −80° C. for subsequent experiments. The bacteria and OMV were viewed by Transmission electron microscopy (SI Appendix).

OMV analysis. A Bradford assay was performed as described previously for quantifying the total protein abundance associated with OMVs. The relative lipid contents of the OMVs were determined via a FM4-64 fluorescence dye binding assay measured by a SpectraMax® iD3 Multi-Mode Microplate Reader (Molecular Devices). The values of the protein amounts and lipid contents were normalized according to the total bacterial number (×1011 CFU). The major outer membrane proteins present in the OMV preparations were detected by immunoblotting. Proteomic analysis of OMVs (SI Appendix).

Animal experiments. All animal studies were conducted in accordance with the NIH “Guide for the Care and Use of the laboratory Animals” and approved by the Institutional Animal Care and Use Committee at Albany Medical College (IACUC protocol #18-02004). Six-week-old male and female Swiss Webster mice were purchased from Charles River Laboratories (Wilmington, MA) and acclimated for one week after arrival. The groups of mice were intramuscularly (i.m.) immunized with 400 μg OMVs in 100 μl PBS buffer, 100 μl of a mixture containing 10 μg LcrV/alhydrogel, as a positive control, or 100 μl PBS/alhydrogel, as a negative control. Booster vaccinations were then administered 3 weeks after the initial vaccination. Blood was collected via submandibular veins every 2 weeks to harvest sera for antibody analysis. At 42 days after the initial vaccination, animals were challenged s.c. withY. pestisKIM6+(pCD1Ap) in 100 μl PBS to mimic bubonic plague. For mimicking pneumonic plague, animals were anesthetized with a 1:5 xylazine/ketamine mixture and were challenged i.n. with virulentY. pestisin 40 μl PBS. The LD50 values ofY. pestisKIM6+(pCD1Ap) administered by s.c. and i.n. challenge in mice were 10 CFU and 100 CFU, respectively. All infected animals were observed over a 15-day period. For the determination of the bacterial burden, the animals were euthanized with an overdose of sodium pentobarbital. Lungs, livers and spleens were removed at the indicated times and homogenized in ice-cold PBS (pH 7.4) using a bullet blender (Bullet Blender Blue; N.Y., USA) at power 7 for 2 min. Serial dilutions of each organ homogenate were plated on HIB agar, and each count was confirmed with duplicate plates with a minimum of 2 dilutions to determine the titers of bacteria per gram of tissue. The experiments were performed twice, and the data were combined for analysis.

Measurement of antibody responses and cytokines. An enzyme-linked immunosorbent assay (ELISA) was used to assay antibody titers against LcrV, F1 orY. pestiswhole cell lysates (YPL) in serum as described in our previous report. A mouse multiplex cytokine assay kit (Bio-Plex) was used to detect the cytokines and chemokines in the BALF and sera collected from the mice according to the manufacturer's instructions.

Analysis of cellular immune responses. Lungs and spleens were obtained aseptically from euthanized animals and dissociated with 70 μm strainers to obtain single cells. The RBC-lysed individual cell populations (2×106) were seeded in 12-well cell culture plates and stimulated in vitro for 72 h with 20 μg/ml rLcrV. Four hours before the collection of the cells, the culture media in each well was supplemented with brefeldin-A and a monensin cocktail (1:1 ratio) to block Golgi-mediated cytokine secretion. For the flow cytometric analysis of the T-cell populations and their corresponding cytokines, the induced cells were harvested and resuspended in FACS staining buffer containing CD16/32 antibodies (1:200) for 10 min on ice. The T-cell-specific markers were stained using anti-mouse antibodies as in Table 3 according to the manufacturer's protocol. The samples were acquired on BD flow cytometers (LSRII) and were analyzed using FlowJo v.10.

TABLE 3Antibodies used in flow cytometry experiments are listed belowAntibodyFlurophoreDilutionCompanyCloneCD3FITC1:200BioLegend17A2CD4PE1:200BioLegendGK1.5CD8APC1:200BioLegendYTS156.7.7IFN-γPerCP Cy5.51:200BioLegendXMG1.2TNF-αHV5101:200BioLegendMP6-XT22IL-2PECy71:200BioLegendJES6-5H4IL17AAPC-Cy71:200BioLegendTC11-18H10.1CD45FITC1:200BioLegend30-F11CD11bHV5101:200BioLegendM1/70CD11cAPC/Cy71:200BioLegendN418Ly6GPE-Cy71:200BioLegend1A8Siglec-FAPC1:200BioLegendS170072F4/80Pacific Blue1:200BioLegendBM8

Cells from the BALF and lungs of mice were resuspended in 30 μL of FACS straining solution containing Fc block (CD16/32) at a 1:100 dilution and incubated at room temperature for 15 min to block macrophage Fc receptors. The cell suspensions were then pelleted at 650×g for five min at 4° C. The cells were resuspended and incubated for 30 min at 4° C. with the following fluorescently labeled antibodies (SI Appendix, Table 3) in flow cytometry buffer (1% BSA in PBS) for the staining of cell surface markers. The stained cells were analyzed based on fluorescence staining patterns to identify the alveolar macrophages (Siglec-F+F4/80+CD11bmid/low+CD11chigh+Ly6G−), monocytes (CD11bhigh+CD11clow+Ly−6G−) and neutrophils (CD45+ Ly−6G+).

Statistical analysis. Each experiment included a significant number (minimum of 3) of biological replicates, with 2-3 replicates performed in a synchronized fashion to establish reproducibility. The statistical analyses of the data among the groups were performed with one-way ANOVA/univariate or two-way ANOVA with Tukey post hoc tests. The log-rank (Mantel-Cox) test was used for the survival analysis. All data were analyzed using GraphPad PRISM 8.0 software. The data are represented as the mean±standard deviation; ns, no significance, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Results

Lipid A 1-dephosphorylation ofY. pestisaffects bacterial morphology and increases OMV biogenesis. Previously,Y. pestisKIM6+ isogenic mutants: χ10015 (ΔlpxP:: PlpxLlpxL) and χ10027 (ΔlpxP:: PlpxL lpxL ΔlacZ:: Plpp lpxE) (Table 1), produced conventional hexa-acylated lipid A and 1-dephosphorylated hexa-acylated lipid A at 28° C. and 37° C., respectively. χ10027 was more susceptible to polymyxin B than KIM6+ and χ10015, suggesting that lipid A 1-dephosphorylation might influence bacterial membrane stability and morphology. Thus, transmission electron microscopy was employed to visualize all three strains when they were cultured in heart infusion broth (HIB) at 28° C. for 14 h and then incubated at 37° C. for 4 h. The morphologies of KIM6+ and χ10015 were observed as a mixture of coccus and bacillus shapes (FIGS.1Aand B), while the χ10027 strain had been completely altered into cocci (FIG.1C). The χ10027 strain had a higher percentage of cell wall bulges that were localized to the bacterial surface than the other two strains (FIG.1).

To determine the effect of lipid A remodeling onY. pestisOMV biogenesis, it was initially confirmed that OMV biogenesis occurred in eachY. pestisstrain cultured at 28° C. for 14 h and then incubated at 37° C. for 4 h. The results showed that KIM6+, ×10015 and χ10027 all produced OMVs, but the sizes of the OMVs from χ10027 were much smaller than those fromY. pestisKIM6+ and χ10015 (FIG.2AandFIG.9). Proteomic analysis by mass spectrometry showed that 293 proteins were detectable in OMVs from all three strains (Table 1) and included 12.3% outer membrane proteins, 4.1% periplasmic proteins and 83.6% cytoplasmic proteins (FIG.2B). OMVs from all three strains contained specific major outer membrane proteins, such as Pla, Ymt, Ail (OmpX), OmpA, F1 and Psn (Table 1). The total protein amounts and lipid contents in OMVs from χ10027 were ˜16- and ˜150-fold increase in comparison to those from KIM6+ and χ10015, respectively (FIG.2C). OMVs from KIM6+ and χ10015 showed comparable total protein amounts and lipid contents (FIG.2C). The amounts of several outer membrane proteins (Psn, OmpA, Pla and F1) were comparable among OMVs isolated from KIM6+, ×10015 and χ10027 (FIG.2D), but the total protein amounts in OMVs from χ10027 were clearly higher than those from KIM6+ and χ10015 (FIG.2E). Thus, the results suggested that lipid A 1-dephosphorylation in χ10027 increased OMV biogenesis, while lipid A acylation in χ10015 did not.

A balanced-lethal system for oversynthesizing LcrV antigen inY. pestis. The three above-described strains harboring the virulence plasmid pCD1 are Select agents and must be studied in a Biosafety Level 3 (BSL3) lab. Growing large cultures of these bacteria in BSL3 for OMV isolation is inconvenient and prohibited. A suite of virulence effectors, Yops (YopE, YopJ, YopH, YopM and YopT), that are encoded on the virulence plasmid pCD1 (˜70 kb) suppress innate immunity to favorY. pestisinfection upon translocation into host mammalian cells by the T3SS. As a vaccine, OMVs derived from pCD1+Y. pestisthat package Yops may result in potential immune suppression. To avoid these concerns, pCD1-deficientY. pestisstrains were used to produce OMVs in a BSL2 lab. However, OMVs from pCD1-deficientY. pestislack the indispensable protective antigen LcrV, which is encoded on the pCD1 plasmid. To overcome this deficiency, a balanced lethal system was constructed to introduce an asd mutation into eachY. pestisstrain to generate YPS1, YPS2 and YPS3, respectively (Table 1), which can adopt an Asd+ plasmid for the oversynthesis of LcrV.

Two Asd+ plasmids were constructed: pSMV12 (V), containing the native lcrV gene ofY. pestisand pSMV13 (Bla-V), containing the N-terminal β-lactamase signal sequence (bla ss) fused withY. pestislcrV to facilitate LcrV secretion into the periplasm by the Type II secretion system (T2SS) (FIG.3Aand Table 1). Subsequently, both plasmids were introduced individually into the YPS1, YPS2 and YPS3 strains to compare the amounts of LcrV in the bacterial cell fractions, including the whole cell lysate, cytoplasm, periplasm, and OMV fractions. The results showed that all mutant strains harboring the Bla-V plasmid secreted more LcrV into the periplasmic fractions than those harboring the V plasmid, indicating that the β-lactamase secretion signal peptide can facilitate LcrV secretion into the periplasmic space inY. pestismutants (FIG.3B). The amounts of LcrV in the cytoplasm and whole cell lysates of each strain harboring the V or Bla-V plasmid were comparable (FIG.3B). Moreover, OMVs isolated from all strains harboring Bla-V enclosed higher amounts of LcrV than those harboring the V plasmid (FIG.3C). Therefore, the pSMV13 (Bla-V) plasmid was chosen for the following studies.

Elimination of potential virulence factors fromY. pestisOMVs.Y. pestisharbors two additional plasmids, pPCP1 (9.6 kb), encoding the plasminogen activator (Pla), and pMT1 (102 kb), encoding murine toxin (Ymt) and the protective antigen F1. Pla is necessary forY. pestisdissemination and the inhibition of immune cell recruitment and induces fibrinolysis. Murine toxin, which is encoded by ymt, is highly toxic in mice and rats but is less toxic in larger animals. Pla and Ymt are clearly present inY. pestisOMVs (FIG.2Dand Table 1). To eliminate the potential adverse effects of Pla and Ymt on hosts, the pPCP1 plasmid was cured and the ymt gene deleted from strains YPS1, YPS2 and YPS3 individually by using sequential steps to generate mutant strains designated YPS7, YPS8 and YPS9, respectively (Table 1,FIG.10). Then, the Bla-V plasmid was individually introduced into the YPS7, YPS8 and YPS9 strains and compared the OMV production of these mutant strains. The results showed that YPS9(Bla-V) with 1-phosphorylated lipid A still generated higher numbers of OMVs (FIG.3D) and enclosed substantially higher levels of LcrV and Psn antigens than YPS7(Bla-V) or YPS8(Bla-V). The amounts of F1 antigen in OMVs derived from each strain were comparable (FIG.3E). Additionally, the total protein amounts in OMVs from YPS9(Bla-V) were clearly higher than those in OMVs from the other two strains (FIG.3F).

The stimulation and cytotoxicity of OMVs from YPS7(Bla-V), YPS8(Bla-V) or YPS9(Bla-V) cultured with different cell lines was compared in vitro and found that OMVs from YPS9(Bla-V) could activate the TLR4-mediated NF-κB signaling pathway but showed less stimulatory activity than OMVs from the other two strains (FIG.11). OMVs from YPS7(Bla-V) or YPS8(Bla-V) at a concentration of 25 μg/ml generated significantly higher amounts of TNF-α and cytotoxicity in RAW 264.7 cells than OMVs from YPS9(Bla-V) at the same concentration, but a low concentration (10 μg/ml) of all three types of OMVs produce decreased amounts of TNF-α without any difference and showed diminished cytotoxicity in cells (FIG.11B). Given the above results, the YPS9(Bla-V) strain was used to produce the greatest amounts of OMVs decorated with MPLA, presenting low toxicity and enclosing high amounts of protective antigens for vaccine evaluation.

Immunization with self-adjuvanting OMVs afforded complete protection againstY. pestischallenge. Groups of mice (n=10) were intramuscularly immunized with OMVs purified from YPS9(Bla-V), LcrV/alhydrogel or PBS/alhydrogel (sham) and boosted at 21 days after the prime immunization (FIG.4A). None of the vaccinations affected body weight increases in mice (FIG.12A) or caused observable health issues. The measurement of serum antibody responses showed that the total anti-LcrV IgG titers were primed at higher levels in LcrV-immunized mice than in OMV-immunized mice at week 2 post vaccination but were boosted to the same levels in both immunized groups by week 4 post vaccination (FIG.4B). The anti-YPL IgG titers (Y. pestiswhole cell lysate) in OMV-immunized animals at weeks 2 and 4 post vaccination were substantially higher than those in LcrV-immunized animals (FIG.4B), also high anti-F1 IgG titers were primed at week 2 post vaccination and boosted at week 4 post vaccination in the OMV-immunized mice (FIG.4B). Additionally, significant anti-LcrV, YPL and F1 IgM titers were primed in OMV-immunized mice at week 2 post vaccination, which were substantially increased at week 4 post vaccination after the booster. The LcrV and YPL IgM titers in the LcrV-immunized mice were not substantially different, but they were significantly lower than those in the OMV-immunized mice at weeks 2 and 4 post vaccination (FIG.12B).

On day 42 after the initial vaccination, the mice were challenged by the subcutaneous (s.c.) or intranasal (i.n.) route to mimic bubonic or pneumonic plague, respectively. All OMV-immunized mice survived s.c. challenge with 8×105 CFU (8×104LD50) ofY. pestisKIM6+(pCD1Ap), while 80% of the LcrV-immunized mice survived the same challenge (FIG.4C). The OMV vaccination afforded 100% and 50% protection in mice against i.n. challenge with a median dose of 5×103 (50 LD50) and a high dose of 5×104 (500 LD50) ofY. pestisKIM6+(pCD1Ap), respectively. The LcrV vaccination conferred decreased protection (FIG.4D), and none of the sham mice survived both challenges (FIGS.4Cand D).

Vaccination with self-adjuvanting OMVs elicited Th1/Th2-balanced immune responses. In mice, IgG1 is associated with a Th2-like response, while a Th1 response is associated with the production of IgG2a, IgG2b, and IgG3 antibodies. Therefore, the IgG subtypes produced in response to each antigen were analyzed to distinguish between Th1/Th2 immune responses in immunized mice. The anti-LcrV IgG1 titers were high and showed similar profiles as the anti-LcrV total IgG titers in both LcrV- and OMV-immunized mice (FIG.4BandFIG.5A). InFIG.5A, it is shown that both LcrV- and OMV-immunized animals were primed with moderate titers of anti-LcrV IgG2a and IgG2b and did not show a substantial difference at week 2 post vaccination. After the booster, the titers of anti-LcrV IgG2a and IgG2b were slightly increased in the LcrV-immunized groups at week 4 post-initial vaccination but rapidly increased in the OMV-immunized groups, and they were significantly higher than those in the LcrV-immunized groups. At week 4 post vaccination, the ratios of anti-LcrV IgG1/IgG2a and IgG1/IgG2b were 1.5 and 1.4 in LcrV-immunized mice, respectively, while the ratios of anti-LcrV IgG1/IgG2a and IgG1/IgG2b were 1.0 and 0.99 in OMV-immunized mice, respectively (FIG.5A). As shown inFIG.5B, the OMV-immunized mice were primed with high titers of anti-YPL IgG1, IgG2a and IgG2b at week 2 post vaccination, which were substantially increased at week 4 post vaccination after the booster. However, the titers of anti-YPL IgG1, IgG2a and IgG2b remained steady in the LcrV-immunized groups at weeks 2 and 4 post vaccination. At week 4 post vaccination, the ratios of anti-YPL IgG1/IgG2a and IgG1/IgG2b were 1.6 and 1.4 in the LcrV-immunized groups, respectively, whereas the ratios of anti-YPL IgG1/IgG2a and IgG1/IgG2b were 1.0 and 1.1 in the OMV-immunized groups, respectively (FIG.5B). Additionally, the OMV-immunized mice were primed with high titers of anti-F1 IgG1, IgG2a and IgG2b at week 2 post vaccination, which were substantially increased at week 4 post vaccination after the booster (FIG.5C). The ratios of anti-F1 IgG1/IgG2a and IgG1/IgG2b were 1.2 and 1.0 at week 4 post-initial vaccination, respectively. Collectively, the OMV-immunized mice generated more broad antibody responses against multiple antigens and more balanced Th1/Th2 responses than the LcrV-immunized mice.

Vaccination with self-adjuvanting OMVs induced potent cellular immune responses. After 72 h of in vitro induction with LcrV or PBS, lung lymphocytes from OMV-immunized mice showed substantial increases in both the CD4 and CD8 T cell populations (FIGS.6A&B). Lung CD4+ T-cells from OMV-immunized mice displayed significantly higher production of IFN-γ, IL-2 and TNF-α than those from LcrV-immunized and sham mice (FIG.6A). Lung CD8+ T-cells from OMV-immunized mice stimulated with LcrV protein showed higher production of TNF-α than those from LcrV-immunized and sham mice, but did not show increased production of IFN-γ, IL-2 and IL-17 in comparison to that in sham- or LcrV-immunized animals (FIG.6B). Both lung CD4+ and CD8+ T-cells from LcrV-immunized mice demonstrated higher production of IL-4 than those from OMV-immunized mice after in vitro stimulation with LcrV (FIGS.6A& B).

Similarly, splenocytes from both OMV- and LcrV-immunized mice also showed increased production of CD4+ and CD8+ T cells in comparison to those from sham mice after in vitro stimulation with LcrV (FIG.13). Spleen CD4+ T-cells from OMV-immunized mice demonstrated significantly higher production of IL-2 and IL-17 than those from LcrV-immunized and sham mice. Significantly higher production of IFN-γ and IL-4 were observed in spleen CD4+ T-cells from both OMV- and LcrV-immunized mice in comparison to those from sham mice. (FIG.13). Spleen CD8+ T-cells from OMV-immunized mice showed higher production of TNF-α than those from LcrV-immunized and sham mice (SI Appendix, FIG. S5B). However, both spleen CD4+ and CD8+ T-cells from LcrV-immunized mice also produced higher levels of IL-4 than those from OMV-immunized mice (FIG.13). These results suggested that OMV vaccination elicited more potent LcrV-specific cellular immune responses in mice than LcrV vaccination.

In vivo responses afterY. pestispulmonary challenge. Furthermore, bacterial burdens were specifically monitored in different tissues, variations of different cells in lung and bronchoalveolar lavage fluid (BALF), and cytokine production in BALF on day 2 after pulmonaryY. pestischallenge to determine the correlation between animal survival and host responses. On day 2 postinfection, the sham mice were found to have strikingly increasedY. pestistiters (mean 7.8 log 10 CFU/g tissue) in lung and moderate bacterial titers in liver (mean 3.8 log 10 CFU/g tissue) and spleen (mean 2.0 log 10 CFU/g tissue). In the LcrV-immunized mice, the bacterial titers reached moderate levels (mean 3.6 log 10 CFU/g tissue) in the lungs, but the bacteria could not disseminate into the liver and spleen (FIG.7A). NoY. pestistiters were observed in the lungs, livers and spleens of OMV-immunized mice (FIG.7A).

Upon the comparison of immunized mice with or without infection, significant increases in CD4+CD44+ cells were observed in the lungs of LcrV- or OMV-immunized mice after infection (FIG.7B). Moreover, the number of CD4+CD44+cells in the lungs of OMV-immunized mice was significantly higher than that in the lungs of sham or LcrV-immunized mice at day 2 post infection (FIG.7B). There were no substantial differences in CD4+CD44+ cell numbers in sham mice pre infection and post infection (FIG.7B). Slight decreases in CD8+CD44+ cells but no significant differences were observed in the lungs of sham, LcrV- and OMV-immunized mice pre infection and post infection (FIG.14). In the BALF, the numbers of alveolar macrophages (AMϕ) in sham mice with or withoutY. pestisinfection were comparable (FIG.7C), but the numbers of neutrophils were dramatically elevated in sham mice afterY. pestisinfection in comparison to those in noninfected mice (FIG.7D). In contrast, the numbers of AMϕ were significantly increased in LcrV- or OMV-immunized mice on day 2 post infection in comparison to those in immunized mice without infection (FIG.7C), while the numbers of neutrophils did not show substantial differences in LcrV- or OMV-immunized mice pre infection and post infection (FIG.7D). In lung tissues, no obvious alterations in AMϕ numbers were observed in mice with or withoutY. pestisinfection (FIG.7E), but the numbers of lung neutrophils were dramatically increased in sham mice compared with LcrV- or OMV-immunized mice on day 2 post infection (FIG.7F). Slight increases in monocytes but no substantial differences were observed in sham, LcrV or OMV-immunized mice pre infection and post infection (FIGS.14B &14C). Additionally, dramatically increased levels of proinflammatory cytokines (IL-1α IL-1β, IL-6, IL-17 and IFN-γ) and chemokines (G-CSF KC, and MIP-1α) associated with the recruitment of neutrophils were secreted into the BALF of sham mice on day 2 post infection in comparison to the levels in sham mice without infection. However, there were no differences in these cytokines and chemokines in the BALF from LcrV- or OMV-immunized mice between pre infection and post infection (FIG.8). These data showed that LcrV or OMV vaccination rapidly activated CD4+ T memory cells, increased the number of AMϕ in BALF and reduced neutrophil recruitment afterY. pestispulmonary infection, which effectively controlledY. pestisdissemination and cytokine storms that typically lead to the rapid death of mice.

Discussion

Generally, the removal of phosphate groups decreases the overall negative charge of a bacterium, thus reducing the electrostatic interactions of the phosphates in lipid A with cationic antimicrobial peptides and decreasing the susceptibility to polymyxin B, which is a cationic antimicrobial peptide that binds negatively charged phosphate groups in lipid A units in LPS on the bacterial membrane and inserts its hydrophobic tail into the outer membranes of bacteria, causing membrane damage and bacteria killing. The removal of 1-phosphate from the conventional biphosphorylated lipid A inE. coliandSalmonelladecreased their susceptibility to polymyxin B, but the opposite was observed inY. pestis. The possible reasons for this are as follows: 1)Y. pestismasked the phosphate groups with 4-amino-4-deoxy-1-arabinose (1-Ara4N) to reduce the negative charge at its surface using different regulatory strategies that those used bySalmonella;2)Y. pestisnaturally lacks O-antigen because bacteria with the full O-antigen are more resistant to polymyxin B than O-antigen isogenic mutants; 3) lipid A 1-dephosphorylation inY. pestismay cause cation displacement in the outer membrane (OM), resulting in a reduction in OM integrity and an increase in OM permeability and thereby changingY. pestismorphology by increasing the OM curvature (FIG.1C) and OMV formation (FIG.2C). The replacement of acylated fatty acid chains (palmitoleate, C16) inY. pestisKIM6+ with laurate (C12) in the χ10015 (ΔlpxP::PlpxLlpxL) strain did not substantially affect bacterial morphology or OMV production (FIGS.1and2C). However, lipid A alteration via the constitutive expression pagL inS. Typhimuriumto remove the β-hydroxymyristoyl group at position 3 in lipid A significantly increased vesiculation and induced OMV production. Thus, alterations in lipid A acylation at different positions in Kdo2 lipid IVA may produce different outcomes during bacterial membrane vesiculation. Further investigations are needed to dissect this process.

Vaccination with OMVs derived from a wild-typeY. pestisstrain containing very low amounts of LcrV provided very limited protection against plague (unpublished data). An Asd+-based balanced lethalSalmonellasystem was adopted with theY. pestissystem that was successful in overcoming this limitation by oversynthesizing LcrV (FIG.3B). The data demonstrated that the localization of the LcrV protein that was secreted into theY. pestisperiplasm by the T2SS led to the enclosure of high amounts of LcrV by OMVs (FIG.3C); Thus, this strategy would be applicable to the delivery of antigens from other pathogens.

In addition to the production of high titers of IgGs against LcrV, YPL and F1 antigen (FIG.4B) that can synergize with cellular immune responses to defend againstY. pestisinfection, vaccination with OMVs also elicited significantly increased titers of IgM against LcrV, YPL and F1 in mice than vaccination with LcrV (SI Appendix,FIG.12B). IgM has been demonstrated to play a protective role in extracellular and intracellular bacterial infections and to facilitate the removal of foreign pathogens due to its efficient agglutination. In mice, B-1a cells spontaneously maintain steady-state levels of natural IgM, while B-1b cells secrete IgM in response to pathogen encounters or heterologous antigens. Recently, it was shown that the capsular F1 antigen ofY. pestiswas recognized by B1b cells and generated high levels of anti-F1 IgM, which played a significant role in responses to plague challenge. It is speculated that high levels of IgM induced by vaccination with self-adjuvanting OMVs containing capsular F1, LcrV or other antigens fromY. pestismay produce better protection against plague than vaccination with LcrV antigen. Further investigations are needed to fully understand the role of IgM secreted from B1b cells in preventingY. pestisinfection.

Previous studies showed that recombinant, bacterially derived OMVs induced a more balanced Th1/Th2 response. Both LcrV and OMV vaccination elicited the production of significant levels of IgG against LcrV and YPL in mice (FIG.4B), but OMV vaccination induced a more balanced Th1/Th2 immune response than LcrV vaccination (FIGS.5Aand B). Consistent with the antibody responses, both lung and spleen CD4+ T-cells from OMV-immunized mice produced higher levels of Th1 cytokines (IFN-γ, IL-2, IL-17 or TNF-α) and significantly lower amounts of Th2 cytokines (IL-4) than those from LcrV-immunized mice after LcrV stimulation in vitro (FIG.6AandFIG.12A). Studies have shown that protection against plague is known to require humoral immunity and cell-mediated immunity induced by IFN-γ and TNF-α. IL-17 also contributes to cell-mediated defense against pulmonaryY. pestisinfection. The induction of potent Th1 and Th17 cell responses by self-adjuvanting OMV vaccination might be one of the primary reasons it offers better protection against lethal infection byY. pestisthan LcrV vaccination (FIGS.4C&D). The detailed mechanisms underlying protective immunity need to be studied further.

The disease progression of primary pneumonic plague in several animal models is biphasic and consists of a preinflammatory and a pro-inflammatory phase. The early ‘preinflammatory’ phase of the disease (initial 36 h post infection) is characterized by rapidY. pestisreplication in the lungs of mice but an absence of measurable host immune responses or obvious disease symptoms. In contrast, the proinflammatory phase (48 h post infection) is characterized by continuous increases in bacterial titers and dramatic increases in the levels of cytokines (IL-1α IL-1β, IL-6, IFN-γ and IL-17) and chemokines (KC, G-CSF, MIP-1α) accompanied by massive neutrophil influx in the lungs and alveolar spaces, resulting in acute lethal pneumonia. The data showed that the responses in the sham group mice on day 2 post infection (FIG.7) were consistent with previous observations. In contrast, both LcrV and OMV vaccination subverted the progression ofY. pestispulmonary infection in mice, resulting in low or absent bacterial titers in the lungs, spleens and livers (FIG.7A) and significant increases in CD4+CD44+ memory T cells (FIG.7B) and AMq in BALF (FIG.7C), which was not observed in sham mice. The results suggested that the presence of memory CD4+T cells, along with high titers of specific anti-Y. pestisantibodies, might activate AMΦ and enhance their phagocytosis, leading to the rapid elimination of inhaledY. pestis.

InY. pestispulmonary infection, the massive recruitment of mature and immature neutrophils in response to an increasing bacterial burden leads to highly necrotic, lethal pneumonia. This phenomenon occurred in sham mice on day 2 post infection and was characterized by dramatic increases in neutrophils in the BALF and lung (FIGS.7D&F) and high amounts of proinflammatory cytokines and chemokines in the BALF and sera (FIGS.8and13). However, the recruitment of neutrophils (FIGS.7Dand F) and the production of proinflammatory cytokines and chemokines (FIGS.8and13) in both OMV- and LcrV-immunized mice were well controlled. Increasingly, evidence has shown that “trained immunity” mediated by innate immune cells primed by encounters with certain pathogens or molecular patterns associated with pathogens (PAMPs) could achieve broad protection. It is speculated that OMV or LcrV vaccination might endow macrophages, neutrophils and other innate cells in the lung with high expression rates of activation markers that allow these cells to form an organized and protective inflammatory response toY. pestisinfection. Therefore, it is worthwhile to further investigate whether the potent “trained immunity” induced by self-adjuvanting OMVs derived fromY. pestisengineered with an array of PAMPs plays an important protective role againstY. pestisinfection.

The studies showed that protective immunity elicited by self-adjuvanting OMVs derived from engineeredY. pestiswas greater than that elicited by LcrV/alhydrogel, suggesting that OMVs could be utilized as antigen carriers for delivering antigens and adjuvants as part of a promising and effective next generation plague vaccine.

Supplemental Information

Bacterial Culture Conditions

AllE. colistrains were grown routinely at 37° C. in LB broth or LB Agar (Difco).E. colistrain, χ7213, was used to construct suicide vectors and conjugate withY. pestisfor generating mutations.Y. pestisgrown in heart infusion broth (HIB) and Tryptose blood agar (TBA) plates was described previously. Strain construction was performed usingY. pestisKIM6+ derivatives that lack the 70 kb pCD1 plasmid and exempt from Select Agent status and can be handled at BSL-2. Ampicillin at 100 μg/ml or chloramphenicol at 25 μg/ml was supplemented to media, when necessary. Fully virulent strainY. pestisKIM6+ (pCD1Ap) was used for animal challenge under BSL-3/ABSL3 containment.

Molecular and Genetic Procedures

Plasmids and primers used in this study were listed in supplemental Table 1 and Table 2, respectively. The lcrV gene was amplified by a lcrV-1/lcrV-3 primer set from genome ofY. pestisKIM6+(pCD1Ap) and cloned into NcoI and HindIII sites of pYA3342 to generate pSMV12. The lcrV gene was amplified by a lcrV-2/lcrV-3 primer set from genome ofY. pestisKIM6+(pCD1Ap) and cloned into EcoRI and HindIII sites of pYA3493 to generate pSMV13. The Δasd flanking region ofY. pestisKIM6+ was assembled by overlapping PCR using Asd-1/Asd-2 and Asd-3/Asd-4 primer sets and cloned into a suicide vector pRE112 to generate pSMV25. To cure pPCP1 plasmid, the replication origin was amplified by the pPCP1-F/pPCP1-R primer set and cloned into pYA4373 to generate pSMV26. All the plasmids were confirmed by PCR screening and DNA sequencing. The procedures for the sacB-based sucrose counter-selectable suicide vectors used to construct unmarked deletion and/or insertion mutations inY. pestiswere described in a previous report. Successful gene mutations were confirmed by PCR screening.

Bacterial Subcellular Fractionation Analysis

Y. pestisstrains were grown in HIB broth at 28° C. for 14 h and then incubated at 37° C. for 4 h The bacterial cells were collected by centrifugation (10,000×g) for 10 minutes. Periplasmic and cytoplasmic fractions were prepared by a lysozyme-osmotic shock method. Equal volumes of periplasmic, cytoplasmic, and supernatant fractions and total lysate samples was analyzed using Western blotting.

Transmission electron microscopy (TEM). Bacterial cultures were absorbed onto freshly glow-discharged Formvar/carbon-coated copper grids for 10 min. The grids were washed in ddH2O and stained with 1% aqueous uranyl acetate (Ted Pella, Inc., CA) for 1 min. The excess liquid was gently wicked off, and the grids were allowed to air dry. The samples were viewed with a JEOL 1200EX transmission electron microscope (JEOL Peabody, MA) equipped with an AMT 8-megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA). The OMVs were analyzed by TEM as described in previous reports.

Stimulation and Cytotoxicity Assay in Cell Lines

To determine stimulatory activity of OMVs via TLR4, HEK-Blue™ hTLR4 and HEK-Blue™ Null1-v cells (InvivoGen, CA, USA) were maintained at 37° C. with 5% CO2 in DMEM (Gibco BRL, Grand Island, NY, USA) containing 10% FBS supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin and 100 μg/ml Normocin. Cells were seeded at a density of 5×104 cells per well in 96-well tissue culture plates (Costar, Washington, DC) and were stimulated with 20 μl OMVs isolated from different strains (final concentration 10 μg/ml or 25 μg/ml) for 8 h. HEK-Blue™ Null1-v cell and PBS as negative controls. Relative NF-κB activity was determined by measuring the embryonic alkaline phosphatase (SEAP) activity that accumulated the culture media according to the manufacturer's instructions.

Murine macrophage RAW264.7 cells were maintained at 37° C. with 5% CO2 in DMEM (Gibco BRL, Grand Island, NY, USA) containing 10% FBS supplemented with 100 μg/ml penicillin and streptomycin. Cells were seeded at a density of 5×104 cells per well in 96-well tissue culture plates (Costar, Washington, DC) and cultured for 12h, and then were stimulated with OMVs isolated from different strains (final concentration 10 μg/ml or 25 μg/ml). 20 ng/ml of LPS as a positive control. After 24 h, the supernatants from each well were collected for measuring TNF-α secretion using Mouse TNF alpha ELISA Ready-SET-Go! kit (Thermo scientific) and lactate dehydrogenase (LDH) release using a Multitox-Fluor Multiplex Cytotoxicity Assay kit (Promega, Madison, USA) following the manufacturer's instructions. Statistical significance among groups were analyzed by two-way multivariant ANOVA with a Tukey post hoc test. ns, no significance, *, P<0.05; **, P<0.01; ***, P<0.001, ****, P<0.0001.

TABLE 4Y. psedotuberculosisstrains and plasmids used in this studyStrain or PlasmidGenotype or relevant characteristicsY. pseudotuberculosisYptb PB1+Y. pseudotuberculosisPB1+, serotype O:1BYptbS32Cure pYV plasmidYptbS40ΔhmsHFRS425 pYV−YptbS41ΔhmsHFRS425 pYV−ΔlacZ044::cafR-cafM-cafA-caf1YptbS42ΔhmsHFRS425pYV−ΔlacI :: PlpplpxE ΔlacZ::caf1R-caf1M-caf1A-caf1YptbS43Δasd ΔhmsHFRS425ΔlacI :: PlpplpxE pYV− ΔlacZ::caf1R-caf1M-caf1A-caf1YptbS44Δasd ΔtolR ΔhmsHFRS425ΔlacI :: PlpplpxE pYV− ΔlacZ::caf1R-caf1M-caf1A-caf1PlasmidspRE112Suicide vector, Cmr, mob−(RP4)R6K ori, sacBpSMV13The full-length lcrV was cloned into pYA3620pSMV59Plpp- bla ss- pagAopin the pYA3342pSMV60Plpp- bla ss- pagAopinto pSMV13

EXAMPLE 2

Construction of an Asd+ Plasmid Containing Genes Encoding Protective Antigens from BothY. pestisandB. anthracis.

Delivering antigens by T2SS into the periplasm space of bacteria could increase the antigens in lumen of OMVs, significantly increasing antibody responses and protective immunity. As mentioned above, protective antigen (PA) of anthrax toxin encoded by pagA is the primary component of human anthrax vaccine. So, the same strategy was applied to construct an Asd+ plasmid to synthesize and secrete PA ofB. anthracisin the heterologous Yptb strain. The pagA gene fragment removing the N-terminal signal sequence is codon-optimized to favor for expression inY. pseudotuberculosis. In addition, the codon-optimized pagA gene (PAopgene) has mutations to eliminate proteolytic cleavage sites, such as a furin site by replacing RKKR167with SNKE167and a chymotrypsin site via deletion of FF314and a substitution at position 308 (E308D), which enhances the stability of the PA protein in the mammalian host. So, the PAopgene fused with N-terminal β-lactamase signal sequence (bla ss) is cloned downstream from Plpppromoter of an Asd+ plasmid (pSMV59) to facilitate PA secretion into the bacterial periplasmic space, resulting in high amounts of PA encased in OMVs. So, pSMV59 was introduced into YPS9 to determine PA synthesis in whole cell lysate and OMV fractions, result showed that OMVs from YPS9 (pSMV59) encased high amounts of PA antigen (FIG.15A).

Based on results shown inFIGS.3and11A, it is possible to construct a new Asd+ plasmid (designated as pSMV60) (FIG.15B) containing both lcrV ofY. pestisand codon-optimized pagA ofB. anthracisand introduce pSMV60 into YPS9 to allow the mutant strain to synthesize both LcrV and PA. Results showed that OMVs isolated from YPS9(pSMV60) contained huge amounts of PA and LcrV antigen (FIG.15B).

AnY. pseudotuberculosis(Yptb) mutant strain was constructed which robustly produces self-adjuvating and highly immunogenic OMVs to deliver protective antigens from different pathogens. Yptb PB1+ (serotype O:1b) is the closest ancestor ofY. pestis, But Yptb is much less virulent thanY. pestis, can be operated in BSL2 lab, and typically causes enteric diseases in humans and animals. With the exception of two additional plasmids carried byY. pestis(pPCP1 and pMT1), the two species share >95% genetic identity and a common virulence plasmid (pCD1/pYV) with a conserved colinear backbone. Yptb grows much faster thanY. pestisat both 28° C. and 37° C. in HIB media, produces higher amounts of OMVs thanY. pestisin the same culture volumes and is much easier to be genetically manipulated thanY. pestis. Therefore, the Yptb PB1+ strain as an alternative to generate high immunogenic and minimal reactogenic OMVs should be an ideal option to achieve similar OMVs but greatly reduce labor-intensive process. To do so, an Yptb mutant strain was constructed, YPtbS41 (Table 4) that produces MPLA, an adjuvant form Lipid A, cures the virulence plasmid pYV to remove all possible immunomodulation factors (Yops) and incorporates the caf1 operon into chromosome to synthesis F1 antigen as an initial strain.

Increasing Production of OMVs.

It is well established that defects in a range of proteins involved in maintaining the structural integrity of the membrane result in increased vesiculation. InE. coli, the five proteins of the Tol-Pal system is comprised of three inner membrane proteins (TolA, TolQ, and TolR) and a periplasmic protein (TolB), which interact with an outer membrane protein, peptidoglycan-associated lipoprotein (Pal). Disruption of tolR inE. coliandSalmonelladid not significantly compromise the cell envelope and growth, but resulted in high levels of OMVs formation. Also individual disruption of vacJ and yrbE resulted in excessive OMV production inHaemophilus influenza, Vibrio choleraorE. coli. NlpI, a lipoprotein, participates in the balance of peptidoglycan breakdown and synthesis.E. coliΔnlpI exhibits hypervesiculation and an increased OMVs production compared to the otherwise isogenic parental strain, without evident leakage of cytoplasmic proteins.Actinobacillus pleuropneumoniaeΔnlpI has similar occurrences. Homologous genes of tolR, vacJ, yrbE and nlpI inY. pseudotuberculosisstrain are YPTS_1234 (83% amino acid identity), YPTS_2737 (75% amino acid identity), YPTS_3704 (83% amino acid identity), and YPTS_0515 (87% amino acid identity) respectively. Therefore, tolR, vacJ, yrbE and nlpI were deleted from wild-type Yptb PB1+ individually and compare OMVs production of each mutant strain with Yptb PB1+. Results have shown that only the tolR mutant largely increases OMVs production, while the vacJ, yrbE or nlpI mutant dose not heighten OMVs production in comparison with Yptb PB1+ (FIG.16). Taken together, the tolR mutation was introduced on top of YPtbS44 (Table 4) to increase OMVs production.

Construction of a Recombinant Yptb Strain Heterologously Expressing the Gene Cluster for β-(1-3)-Glucan Synthesis.

More and more evidences indicate that plague vaccines aiming to induce mixed Th1 and Th17 cellular responses would provide more powerful and comprehensive protection. Curdlan acts as an adjuvant for the activation of Th1 and, in particular, Th17 immunity. Curdlan is a high-molecular-weight water insoluble β-(1-3)-D-glucan (glucose homo-polymer) without any substituents that has been approved as a food additive by the U. S. FDA. Curdlan is produced by anAgrobacteriumsp. (formerly known asRhizobium lupini) and some other bacteria. Four genes are involved in curdlan biosynthesis (crdA, crdS, crdC and crdR). The curdlan synthase (CrdS), is the key enzyme of curdlan biosynthesis. The UDP-glucose is also a critical block for the curdlan synthesis. So far, there are no any reports about exact UDP-glucose synthesis in Yptb. Protein blast shows that Yptb has a galUF operon governing UDP-glucose synthesis. Therefore, introducing the curdlan synthesis operon (crdASCR) into a certain site of chromosome in YPtbS39 (Table 4) that would synthesize β-(1-3)-glucan will be explored. In addition, studies have shown that production of curdlan is activated by the second messenger c-di-GMP binding to glucan synthase, CrdS. So, the mutant strain combining elevation of c-di-GMP and curdlan synthesis operon would increase curdlan synthesis.

EXAMPLE 3

The PcrV forms a ring structure at the tip of the needle of Type three secretion system (T3SS) in PA and is essential for translocation of the effectors and bacterial pathogenicity. PcrV is a conserved protein among different serotypes of PA isolates and a promising antigen candidate. Immunization with recombinant PcrV or adaptive transfer of anti-PcrV antibodies offered significant protection against lethal PA infections. In addition, iron is an indispensable nutrient for replication of almost all bacteria. Several iron acquisition systems are used by PA to obtain iron from mammalian hosts during infection and play an important role in bacterial virulence. The hitA (PA4687), hitB (PA4688), and others in PA are involved in iron transportation and associated with bacterial virulence. A study showed immunization with ferric iron-binding periplasmic protein HitA afforded protection against PA infection in mice. Therefore, immunization with OMVs delivering heterologous PcrV and HitA antigens as a bivalent vaccine might potentiate protective immunity against PA infection.

Here, an Asd (Aspartate-semialdehyde dehydrogenase)-based balanced-lethal recombinantYersinia pseudotuberculosissystem tailored with an Asd+ plasmid was used to over-synthesize the heterologous PcrV-HitAT fusion antigen (referred to PH), as well as produce high amounts of OMVs encasing the PH antigen. Intramuscular (i.m.) immunization with the rOMV-PH stimulated robust B and T-cell responses and offered great protection against lethal subcutaneous (s.c.) or intranasal (i.n.) challenge with PA103 strain.

Materials And Methods

Bacterial strains, plasmids, culture conditions, and molecular operations. All bacterial strains and plasmids used in this study were listed in the Table 1. All bacterial cultures and molecular and genetic procedures used in this study were described in the Supplementary information (SI).

OMV isolation and analysis. Isolation of OMVs fromY. pseudotuberculosisstrains was similar as described previously. A brief procedure was described in SI. The OMVs were analyzed by Transmission electron microscopy (TEM), a Bradford assay was performed for quantifying the total protein abundance associated with OMVs as described previously. The heterologous antigen present in the OMV preparations were detected by immunoblotting.

Animal experiments. Animal protocols were in accordance with the NIH “Guide for the Care and Use of the laboratory Animals” and were approved by the Institutional Animal Care and Use Committee at Albany Medical College (IACUC protocol #20-02001). Six-week-old male and female BALB/c mice were purchased from Taconic (Germantown, NY) and acclimated for one week after arrival. Mice were primed by intramuscular (i.m.) vaccination, then boosted at 3 weeks after the initial vaccination. Blood samples were collected via submandibular veins at 2-week intervals to harvest sera for antibody analysis. On 42 days after the initial vaccination, animals were challenged subcutaneously (s.c.) with a lethal dose of PA103 strain in 100 μl PBS to mimic surgical infection. For mimicking acute pneumonic infection, animals were anesthetized with a 1:5 xylazine/ketamine mixture and were challenged intranasally (i.n.) with a lethal dose of PA103 in 40 μl PBS. All infected animals were observed over a 15-day period. The actual numbers of bacterial CFUs were determined by plating serial dilutions of the inoculum on LB agar plates.

For the determination of the bacterial burden, infected animals were euthanized with an overdose of sodium pentobarbital. Lungs, livers, spleens and blood were taken at the indicated times and homogenized in ice-cold PBS (pH 7.4) using a bullet blender at power 7 for 2 min. Serial dilutions of each organ homogenate were plated on LB agar plates, and each count was confirmed with duplicate plates with different dilutions to determine the titers of bacteria per gram of tissue. A mouse multiplex cytokine assay kit (Bio-Plex, Bio-rad) was used to detect cytokines and chemokines in the serum and bronchoalveolar lavage fluid (BALF) collected from the mice according to the manufacturer's instructions.

Antibody responses and opsonophagocytic killing assay. Antibody titers were measured using an enzyme-linked immunosorbent assay (ELISA) described in SI. The opsonophagocytic killing assay were performed as described previously. Briefly, HL-60 cells (ATCC, CCL-240) were differentiated into granulocyte-like cells in the Iscove's Modified Dulbecco's Medium (IMDM) (ATCC) containing 100 mM N′,N-dimethylformamide (Sigma) for 5 days. Sera samples from immunized mice containing opsonic antibodies were heat-inactivated (56° C., 30 min) and serially diluted with opsonization buffer (mixture of 80 ml of sterile water, 10 ml of 10× Hank's balanced solution, 10 ml of 1% gelatin, and 5.3 ml of fetal bovine serum). Each well in a 96-well plate contains: 40 μl of 4×105 HL60 cells, 103 CFUs of PA103 in 10 μl of opsonophagocytic buffer, 20 μl of serum, and 10 μl of 1% infant rabbit serum as a complement source (Sigma). Blank wells with the same system in absence of mouse serum were used as negative controls. After 2 h incubation, 10 μl of each sample was plated on LB agar medium. Each sample was performed in triplicate. The opsonophagocytic killing ability was defined as a reduction in CFUs compared with the CFUs in the sera from unimmunized mice.

Analysis of cellular immune responses. Lungs and spleens were obtained aseptically from euthanized animals and lungs were minced and digested with 400 μg/ml of Liberase and 30 μg/ml of DNase (Sigma) at 37° C. for 30 min. Then, tissues were dissociated with 70 μm strainers to obtain single cells. The RBC-lysed individual cell populations (2×106) were seeded in 12-well cell culture plates and stimulated in vitro for 72 h with 20 μg/ml PH. Four hours before the collection of the cells, Cells in each well were supplemented with brefeldin-A and a monensin cocktail (1:1 ratio) to block Golgi-mediated cytokine secretion. For the flow cytometric analysis of the T-cell populations and their corresponding cytokines, the induced cells were harvested and resuspended in FACS staining buffer containing CD16/32 antibodies (1:200) for 10 min on ice. The T-cell-specific markers were stained using anti-mouse CD3 (FITC), CD4 (PE) and CD8 (APC) antibodies (BioLegend, CA), followed by intracellular cytokine (IFN-γ, PerCP Cy5.5; TNF-α, BV510; IL17A, APC-Cy7) staining using BioLegend Perm-fix solution and buffer according to the manufacturer's protocol. The entire staining process was performed on ice with 30 min incubation at each step. The events were acquired on BD flow cytometers (FACSymphony A3) and analyzed using FlowJo v.10.

Statistical analysis. The statistical analyses of the data among the groups were performed with one-way ANOVA/univariate or two-way ANOVA with Tukey post hoc tests. The log-rank (Mantel-Cox) test was used for the survival analysis. All data were analyzed using GraphPad PRISM 8.0 software. The data were represented as the mean±standard deviation (SD); ns, no significance, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Results

OMVs displaying the heterologous PcrV-HitAT fusion antigen ofP. aeruginosa. A hypervesiculating and ΔasdY. pseudotuberculosismutant strain, YptbS44 [Table 1 and Supplementary information (SI)] producing adjuvant monophosphoryl lipid A (MPLA) was used here. YptbS44 adapted an Asd+ plasmid, pSMV81 (Table 1,FIG.17Aand SI), in which the perV-hitAT fusion DNA fragment was ligated with the N-terminal β-lactamase signal sequence (bla ss) to facilitate secretion of PcrV-HitAT (PH) into the bacterial periplasm by Type II secretion system. Determination of the PH synthesis showed that YptbS44 harboring pSMV81 synthesized high amounts of PH (MW, 68 kDa) in bacterial cell lysates (BCL) and produced high amounts of OMVs carrying considerable amounts of PH fusion antigen (FIGS.17Band C). The 10 μg of OMVs from YptbS44(pSMV81) contained around 1 μg PH in comparison to the standard amounts of PH-His fusion protein. No PH was present in YptbS44 harboring an empty plasmid pYA3493 (FIGS.17Band C).

Immunization with OMVs enclosing PH antigen afforded significant protection againstP. aeruginosainfection. Prior to challenge study, it was determined that LD50s (50% of lethal dose) of WT PA103 in BALB/c mice by s.c. and i.n. administration were 7.1×106 colony-forming unit (CFU) and 2×105 CFU, respectively. The LD50s of PA103 were similar to previous report. Groups of mice (n=10-15, nearly equal males and females) were immunized intramuscularly with 100 μl PBS containing 50 μg of OMVs from YptbS44(pSMV81) designated as rOMV-PH, 50 μg of OMVs from YptbS44(pYA3493) designated as rOMV-N, PH (10 μg)/alhydrogel or PBS/alhydrogel, then boosted at 21 days after the prime immunization (FIG.18A). The rOMV-PH immunization slightly slowed down mouse weight gaining in the first week after immunization in comparison to other immunization groups (FIG.18B), but did not cause any observable health issues in mice. On day 42 after the initial vaccination, mice were challenged by s.c. or i.n. route, respectively. All rOMV-PH-immunized mice survived s.c. challenge with 6.7×107 CFU (10 LD50) of PA103, while 50% of the rOMV-N-immunized mice and 40% of PH-immunized mice survived the same challenge (FIG.18C). The rOMV-PH vaccination afforded 73% protection for mice against i.n. challenge with 5×106 (25 LD50) of PA103, only 20% of PH-immunized mice and no rOMV-N-immunized mice survived the same challenge (FIG.18D). None of the PBS-immunized mice survived both challenges (FIGS.18Cand D).

rOMV-PH vaccination elicited strong antibody responses with significant opsonophagocytic killing capability. Serum antibody responses showed that both PH- and rOMV-PH prime immunization generated similarly high anti-PH IgG titers in mice at week 2 post vaccination, and both boost immunization substantially increased anti-PH IgG titers at week 4 post vaccination (FIG.19A). Low unspecific anti-PH IgG titers in rOMV-N-immunized mice were stimulated at both weeks 2 and 4 post vaccination (FIG.19A). Generally, IgG1 is associated with a Th2-like response, while IgG2a is associated with a Th1-like response in mice. Therefore, IgG subtypes to the specific antigen produced in immunized mice can distinguish Th1 and Th2 immune responses. InFIG.19B, both ratios of anti-PH IgG2a/IgG1 in the rOMV-PH-immunized group (0.998 and 0.891) and the rOMV-N-immunized group (1.031 and 1.046) at weeks 2 and 4 post immunization were substantially higher than those in the PH-immunized group (0.673 and 0.668). Our results demonstrated that the rOMV immunization generated a balanced Th1/Th2 responses, while the PH-immunization skewed to Th2-biased response. In addition, compared with the PH or rOMV-N-immunization, the rOMV-PH immunization primed significantly high anti-PH IgM titers in mice at week 2 post vaccination and remained consistent levels at week 4 post vaccination (FIG.19C). Anti-PH IgM titers in the PH-immunized mice were substantially higher than those in the rOMV-N-immunized mice at week 2 post vaccination, but declined to the same levels as those in the rOMV-N-immunized mice at week 4 post vaccination (FIG.19C).

Opsonophagocytic killing (OPK) assay has been used to evaluate correlation of functional antibody levels in serum samples with protection. Thus, whether the PH-specific antibodies were protective was measured using OPK assay. Results showed that only undiluted anti-sera from rOMV-PH-immunized mice exhibited significant OPK activity compared with those from PBS-, PH- or rOMV-N-immunized mice. While, all diluted anti-sera (1:10 or 1:100) from three immunized groups displayed no OPK activity (FIG.19D).

Vaccination with rOMV-PH induced potent cellular immune responses. Following, T-cell responses were evaluated in the lung and spleen after immunization. Lung cells from rOMV-immunized mice showed increased number of CD4+ and CD8+ T cells in comparison to those from PBS- or PH-immunized mice after in vitro stimulation with PH (FIGS.20Eand F). Further, cytokine producing T cells were analyzed by Flow cytometry. Lung CD4+ T-cells from rOMV-PH-immunized mice displayed significantly higher production of IFN-γ, IL-17A or TNF-α than those from PH- and PBS-immunized mice after in vitro induction with PH (FIG.20B). Production of IFN-γ, IL-17A or TNF-α in lung CD4+ T-cells from PH-immunized mice was low in comparison to that from rOMV-PH-immunized mice, but was significantly higher than that from PBS-immunized mice (FIG.20B). Lung CD8+ T cells from rOMV-PH-immunized mice showed comparable IFN-γ production to those from PH-immunized mice, but higher IFN-γ production than those from PBS-immunized mice after stimulation. Comparable levels of IL-17 and TNF-α were produced in rOMV-PH-, PH- or PBS-immunized animals (FIG.20D).

In similar fashion, splenic CD4+ T cells from rOMV-PH-immunized mice significantly increased after in vitro stimulation with the PH antigen in comparison to cells from PBS- or PH-immunized mice. There was no significant increase in splenic CD4+ T cells from PH-immunized mice compared to cells from PBS-immunized mice (FIGS.21Eand F). Splenic CD8+ T cells from rOMV-PH-immunized mice after stimulation were substantially higher than those from PBS- or PH-immunized mice. Also, splenic CD8+ T cells from PH-immunized mice showed increased induction in comparison to those from PBS-immunized mice (FIGS.21Eand F). Specifically, splenic CD4+ T-cells from rOMV-PH-immunized mice demonstrated significantly higher production of IFN-γ, IL-17A or TNF-α than those from PH- and PBS-immunized mice (FIG.21B). Also, higher production of TNF-α was observed in spleen CD4+ T-cells from PH-immunized mice in comparison to those from PBS-immunized mice. (FIG.21B). Spleen CD8+ T cells from rOMV-PH-immunized mice showed higher production of IFN-γ and TNF-α than those from PH- and PBS-immunized mice. Production of IL-17A in splenic CD8+ T cells was low and no significant difference among three immunized groups (FIG.21D). In addition, lung and spleen lymphocytes and their cytokine production were comparable without antigen stimulation in each immunized group (data not shown). Altogether, these results indicated that rOMV-PH vaccination elicited more potent antigen-specific T-cell responses in mice than PH vaccination.

The rOMV-PH vaccination effectively controlled bacteria and host inflammation. Further, in vivo responses of immunized mice challenged with a sub-lethal dose of PA103 were evaluated. Mice were challenged with 5×106 CFU PA103 by s.c. administration and monitored bacterial burdens in different tissues and cytokine/chemokine production in serum on 36 h post infection. Results showed that striking increases of PA titers in lungs (mean 5.5 log 10 CFU/g tissue) and livers (mean 6.2 log 10 CFU/g tissue), and moderate bacterial titers in spleens (mean 4.8 log 10 CFU/g tissue) and blood (mean 3.8 log 10 CFU/g tissue) in the PBS-immunized mice. Bacterial titers within all four organs in PH-immunized mice substantially decreased in comparison to PBS-immunized mice, but still retained significantly higher in lungs and spleens than those in rOMV-PH-immunized mice. No bacteria disseminated to those organs in rOMV-PH-immunized mice (FIG.22A). Analysis of serum cytokine/chemokine in mice on 36 h post s.c. infection showed that dramatically increased levels of cytokines (IL-1β and IL-6) and chemokine (KC) were secreted into the sera of PBS-immunized mice in comparison to those in PH- or rOMV-PH-immunized mice. Levels of serum IL-10 in both PBS- and PH-immunized mice were substantially higher than those in rOMV-PH-immunized mice, but levels of serum IL-10 in PH-immunized mice were even higher than those in PBS-immunized mice (FIG.22B). The rOMV-PH-immunized mice produce the greatest amounts of serum IL-17A among all three immunized groups. In comparison to PBS-immunized mice, PH-immunized mice also produced significantly high amounts of IL-17A. while, levels of TNF-α were comparable in three immunization groups after infection (FIG.22B).

Similarly, groups of immunized mice were evaluated by i.n. challenge with 5×105 CFU PA103. On 36 h post pulmonary infection, PBS-immunized mice were found to have strikingly increased bacterial titers in lungs (mean 7.0 log 10 CFU/g tissue), and rapidly disseminated to livers (mean 6.0 log 10 CFU/g tissue), spleens (mean 5.8 log 10 CFU/g tissue) and blood (mean 4.8 log 10 CFU/g tissue). In comparison to the PBS immunization, the PH- or OMV-PH immunization substantially decreased bacterial burdens within lungs, livers and spleens of mice. However, the OMV-PH immunization had more efficiency to clear bacteria from mouse blood than the PH-immunization (FIG.23A). Analysis of BALF cytokine/chemokine in mice after i.n. infection showed that dramatically high levels of cytokines IL-6, IL-10, IFN-γ and TNF-α) and chemokine (KC) were secreted into the BALF of PBS-immunized mice on 36 h post infection in comparison to those in PH- or rOMV-PH-immunized mice (FIG.23B). The rOMV-PH-immunized mice produced even less amounts of IL-10 and IL-6 than the PH-immunized mice did. Like serum cytokine responses, the rOMV-PH-immunized mice produced significantly higher amounts of IL-17A in BALFs than PBS- or PH-immunized mice did (FIG.23B).

Discussion

The increasing prevalence of multidrug-resistantP. aeruginosa(PA) infections in healthcare settings justifies the urgent need for an effective vaccine against this organism. Barriers to PA vaccine development include the presence of phenotypically diverse PA strains, the diverse virulence mechanisms, and lack of reliable animal models to mimic CF patients. A number of PA vaccine candidates are being tested in clinical trials, but so far no licensed vaccines are available for human use. Among them, a PA subunit vaccine (IC43) composed of OprI and a fragment of the outer membrane protein OprF was evaluated in a phase III clinical trial (NCT01563263). Immunization with 100 μg of IC43 was well tolerated in a large group of mechanically ventilated patients as well as achieved high immunogenicity, but did not present significant clinical benefit over placebo in terms of overall mortality. Human clinical trials showed that anti-PcrV antibody or its fragment could reduce inflammation and damage of the airway of CF patients, but directly using PcrV antigen as a vaccine component seemed to never be evaluated in human clinical trials probably due to protein purity or other unmentioned issues. In addition, Holder et al reported that immunization with PcrV alone did not provide long-term protection to burned mice infected with the highly toxigenic strain 1071. Moreover, purified antigens as subunit vaccines administered alone have limited immunogenicity and vaccination with subunit vaccines prefers to generate humoral response. Many studies reach consistent points that an excellent PA vaccine should stimulate antibodies combined with both Th1- and Th17-type CD4+ T cell responses to provide effective protection against pulmonary and systemic PA infection. Currently, this dilemma for subunit vaccines is being addressed with different improved vaccine carriers. Among them, using self-adjuvanting OMVs as a carrier not only circumvents the requirements of antigen-purification for traditional subunit vaccines, but also stimulates potent specific humoral and cellular responses to the delivered antigens.

Our studies showed that i.m. immunization with rOMV-PH afforded complete protection against s.c. challenge and 73% protection against i.n. challenge with the virulent PA103 strain (FIGS.18Cand D), providing evidence that using rOMVs from a recombinantY. pseudotuberculosisstrain to deliver a heterologous PH fusion antigen ofP. aeruginosawas feasible. Less protection against pneumonic infection might be owing to the immunization route. Usually, systemic immunization like i.m, or s.c. is less effective in generation of high-avidity natural antibodies or cellular responses at mucosal sites and results in inferior protection from mucosal challenge. Thus, combining prime-boost immunization with systemic and mucosal rOMV-PH might achieve complete protection against both systemic and respiratory PA infection. In addition, combination of triple- or tetra-antigen may further enhance protection against PA infection.

Generally, antigen-specific antibodies induced by a vaccine candidate are able to correlate with protection and assist killing of host target cells infected by bacteria. However, our results showed that immunization with the PH/alhydrogel generated comparable high titers of PH-specific antibody in mice to the rOMV-PH immunization (FIG.19A) but failed to provide effective protection against s.c. or i.n. PA103 challenge (FIGS.18Cand D). A possible reason is the PH immunization stimulated Th2-biased immune response (IgG2a/IgG1=0.67), while rOMV-PH vaccination induced more balanced Th1/Th2 immune response (IgG2a/IgG1=0.9) (FIG.19B). In addition, the rOMV-PH immunization primed significantly higher anti-PH IgM titers in mice than the PH-immunization at weeks 2 and 4 post vaccination (FIG.19C). A growing evidence indicates that IgM provides a first line of defense during microbial infections, prior to the generation of high affinity IgG responses. However, whether the antigen-specific IgM is involved in the protection needs to be further investigated in the future. Interestingly, in vitro OPK assay showed that anti-sera from rOMV-PH immunized mice could effectively kill PA, instead of anti-sera from PH-immunized mice (FIG.19D). Our results were not consistent with several previous studies, in which anti-PcrVNH sera from the PcrVNH-immunized mice or POH-specific antibodies from the trivalent subunit (PcrV-OprI-Hcp1, POH) vaccinated mice exhibited significant OPK activity to PA. Regarding these, it is speculated that distinct antigen combination or probably different antibody compositions in sera from immunized mice may lead to the inconsistency.

Based upon antibody analysis (FIG.19), humoral response alone could not provide optimal protection against PA infection, while cellular immune responses are required to achieve comprehensive protection. Measurement of T cell responses in lungs and spleens showed that the rOMV-PH immunization stimulated substantially higher T cell responses than the PH immunization (FIGS.20and21), which is well correlated with animal survival after challenge (FIGS.18Cand D). In addition, consistent with the immune protection and responses, rOMV-PH-immunized mice had more efficiency to clear PA after s.c. or i.n. challenge than the PH- or PBS-immunized mice (FIGS.22A and23A). Measurement of cytokines/chemokines in the sera and BALFs of mice on 36 h post PA103 infection indicated that the rOMV-PH immunization more effectively coordinated cytokines/chemokines production in mice than the PH or PBS immunization (FIGS.22B and23B). Studies have implicated that overproduction of IL-10, IL-6 and IL-10 in mice and human are associated with sepsis and IFN-γ expression was enhanced persistently in patients who died of sepsis. In consistence to mouse survival after s.c. PA103 challenge, the amounts of serum IL-1β, IL-6 IL-10, and IFN-γ in PH-immunized mice were significantly higher than those in rOMV-PH-immunized mice on 36 h post infection (FIG.22B). Jin et al showed that CXCL1 (KC) contributed to host defense in polymicrobial sepsis. However, our data showed that rOMV-PH-immunized mice produce less amounts of KC in sera than PH-immunized mice after infection (FIG.22B). In addition, KC is essential for neutrophil migration and expression of proinflammatory mediators, thus overmounts of KC in sera recruiting excessive neutrophils may cause tissue damage and organ failure. In similar manner, higher amounts of IL-1β and IL-6 secreted into BALFs of PH-immunized mice after i.n. PA103 challenge than those of rOMV-PH-immunized mice, which can reflect animal survival after infection (FIG.23B). However, rOMV-PH-immunized mice produced substantially higher IL-17 in both sera and BALFs than PH-immunized mice after both s.c. and i.n. challenge (FIGS.22B and23B), suggesting that IL-17 plays a protective role against PA infection. Our results were consistent to several previous studies, but the detailed role of IL-17 induced by the rOMV-PH immunization for protection needs to be pursued further. Altogether, our studies showed that self-adjuvating OMVs from recombinantY. pseudotuberculosiscould be as a carrier for delivering a heterologous fusion antigen (PH) and vaccination with rOMV-PH induced robust humoral, Th1 and Th17 cell responses, resulting in comprehensive protection against lethal PA infection. Thus, OMVs delivering PA antigens would be a novel promising vaccine preventing unrestrained spread of PA in healthcare settings.

EXAMPLE 4

TABLE 5P. aeruginosastrains and plasmids used in this studyStrain or PlasmidGenotype or relevant characteristicsStrainsE. coliTop 10F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupGχ6212F- λ- ϕ80 Δ(lacZYA-argF) endA1 recA1 hsdR17 deoR thi-1 glnV44gyrA96 relA1 ΔasdA4SM10(λpir)Kmr; thi-1 thr-1 leuB26 tonA21 lacY1 supE44 recA integrated RP4-2 Tcr::Mu aphA+(RP4-2 is RP4 ΔTn1)RH03Kms; Δasd::FRT ΔaphA::FRT SM10(λpir)P. aeruginosaP. aeruginosaPA103Wild-type strainΔexoU PA103ΔexoUPA-m1ΔlpxL1PA-m2ΔexoU ΔwbjAPA-m3ΔexoU ΔwbjA ΔexoAPA-m4ΔexoU ΔwbjA ΔexoA ΔexoTPA-m5ΔexoU ΔwbjA ΔexoA ΔexoT ΔlasAPA-m6ΔexoUΔwbjAΔexoAΔexoTΔlasAΔlasBPA-m7ΔexoUΔwbjAΔexoAΔexoTΔlasAΔlasBΔpchAPA-m8ΔexoUΔwbjAΔexoAΔexoTΔlasAΔlasBΔpchAΔphzMPA-m9ΔexoUΔwbjAΔexoAΔexoTΔlasAΔlasBΔpchAΔphzMΔalgPA-m10ΔexoUΔwbjAΔexoAΔexoTΔlasAΔlasBΔpchAΔphzMΔalgΔRhlABPA-m11ΔexoUΔwbjAΔexoAΔexoTΔlasAΔlasBΔpchAΔphzMΔalgΔRhlABΔpvdAPA-m12ΔexoUΔwbjAΔexoAΔexoTΔlasAΔlasBΔpchAΔphzMΔalgΔRhlABΔpvdAΔplcHPA-m13ΔexoUΔwbjAΔexoAΔexoTΔlasAΔlasBΔpchAΔphzMΔalgΔRhlABΔpvdAΔplcHΔlpxLPA-m14ΔexoUΔwbjAΔexoAΔexoTΔlasAΔlasBΔpchAΔphzMΔalgΔRhlABΔpvdAΔplcHΔlpxLΔphoAPlasmidspYA3342Asd+vector, Ptrc,pBR oripYA3493Asd+vector with β-lactamase N-terminal signal sequence, Ptrc,pBRoripDMS197Suicide vector, Tetr, mob−(RP4)R6K ori, sacBpUCP20E. coli-Pseudomonasshuttle vector; AprCbrpSMV81The pcrV-hitATDNA fragment was cloned into sites of EcoRI andHindIII in the pYA3494pSMV82The pcrV-hitAT−6xhis fragment was cloned into sites of NcoI andHindIII in the pYA3342pSMV83The Ptrc-bla ss-pcrV-hitATDNA fragment was cloned into thepUCP20

TABLE 6Primers used in theP. aeruginosastudy.Primer nameSequencea(5′ to 3′)exoT-UFcgggagctctatccatcgggttctccgccccgg(SEQ ID NO: 10)exoT-URtggcaacgccggggtcccgggaggggcaggcggcgcgtcctgacggga (SEQ ID NO: 11)exoT-DFtcccgtcaggacgcgccgcctgcccctcccgggaccccggcgttgcca (SEQ ID NO: 12)exoT-DRcggtctagatgactgcgtctcgttcg(SEQ ID NO: 13)exoA-UFcgggagctcgacagctcggcgtagaccagc(SEQ ID NO: 14)exoA-URacccatcacaggagccatcgcggtggtgattccctcggcgatc (SEQ ID NO: 15)exoA-DFgatcgccgagggaatcaccaccgcgatggctcctgtgatgggt (SEQ ID NO: 16)exoA-DRcggtctagagcgacgctcgacaatgctct(SEQ ID NO: 17)lasA-UFcgggagctcgtcggcggcttcttcgggccgc(SEQ ID NO: 18)lasA-URttcgatgaccaggagctacccgtcggcgcggggcccggctcca (SEQ ID NO: 19)lasA-DFtggagccgggccccgcgccgacgggtagctcctggtcatcgaa (SEQ ID NO: 20)lasA-DRcggtctagaagccggacgaggacgacggtta(SEQ ID NO: 21)lasB-UFcgggagctcgatgttccacggggtgttcca(SEQ ID NO: 22)lasB-URtgctggccggggccaccgagcttacttgttcagttctcctggttttttc (SEQ ID NO: 23)lasB-DFgaaaaaaccaggagaactgaacaagtaagctcggtggccccggccagca (SEQ ID NO: 24)lasB-DRcggtctagaggtcgtgtgctggggatcgaa(SEQ ID NO: 25)wbjA-UFcgggagctcgctgctacttcacccatagctagcg(SEQ ID NO: 26)wbjA-URctttctatcgagaacccccttccagactgcgctacaaggccggccagga (SEQ ID NO: 27)wbjA-DFtcctggccggccttgtagcgcagtctggaagggggttctcgatagaaag (SEQ ID NO: 28)wbjA-DRcggtctagacccaccataacaccatatgcggtca(SEQ ID NO: 29)pchA-UFcgggagctccacctgttcgtctccgcccatc(SEQ ID NO: 30)pchA-URggccgcagggggtcttcgtttgcggcaccccgtgtctggcgc (SEQ ID NO: 31)pchA-DFgcgccagacacggggtgccgcaaacgaagaccccctgcggcc (SEQ ID NO: 32)pchA-DRcggtctagaaactaatcgccatgaatgaaaa(SEQ ID NO: 33)phzM-UFcgggagctcgctgccggaggacgtggagaac(SEQ ID NO: 34)phzM-URtggccttcgagatctttcagggatcggaactctcaacggttggc (SEQ ID NO: 35)phzM-DFgccaaccgttgagagttccgatccctgaaagatctcgaaggcca (SEQ ID NO: 36)phzM-DRcggtctagaaaggcaataggagtttcatccag(SEQ ID NO: 37)alg-UFcgggagctcgacgtgctgctcaacctggcttcc(SEQ ID NO: 38)alg-URcatcttcatggtcgggtaccggtaggatgttttctctgcgaggg (SEQ ID NO: 39)alg-DFccctcgcagagaaaacatcctaccggtacccgaccatgaagatg (SEQ ID NO: 40)alg-DRcggtctagacgccctggtcgggatagtcgta(SEQ ID NO: 41)rhlAB-UFcgggagctcctgcctgggcaagagcacctac(SEQ ID NO: 42)rhlAB-URtatctgttatgccagcaccgtttcacacctcccaaaaatttt (SEQ ID NO: 43)rhlAB-DFaaaatttttgggaggtgtgaaacggtgctggcataacagata (SEQ ID NO: 44)rhlAB-DRcggtctagaggcgatttccccggaactcttg(SEQ ID NO: 45)pvdA-UFcgggagctctggaacgcctgctcgccgctca(SEQ ID NO: 46)pvdA-URgccaatccagaggaactggaatcggcgccacgccgccacgc (SEQ ID NO: 47)pvdA-DFgcgtggcggcgtggcgccgattccagttcctctggattggc (SEQ ID NO: 48)pvdA-DRcggtctagatgtcttcatcgagggttccagtta(SEQ ID NO: 49)plcH-UFcgggagctcttgacttccggtgggtaggtttcg(SEQ ID NO: 50)plcH-URaccacccgggaaataaaacgagcgaggagtccatcgcatga (SEQ ID NO: 51)plcH-DFtcatgcgatggactcctcgctcgttttatttcccgggtggt (SEQ ID NO: 52)plcH-DRcggtctagaggagtagtggccgatgatccct(SEQ ID NO: 53)htrB2-UFcgggagctcgcgcaccggagtcttcaccacctt(SEQ ID NO: 54)htrB2-URcgcgtccggaatgcccgtccggacggttccgacgacgatca (SEQ ID NO: 55)htrB2-DFtgatcgtcgtcggaaccgtccggacgggcattccggacgcg (SEQ ID NO: 56)htrB2-DRcggtctagatcgccgaagtactcgcggttga(SEQ ID NO: 57)phoA-UFcgggagctcctgtgcaaattgttgcgcacat(SEQ ID NO: 58)phoA-URcctttttcgttctggtccgagacgcatttccctatgttgag (SEQ ID NO: 59)phoA-DFctcaacatagggaaatgcgtctcggaccagaacgaaaaagg (SEQ ID NO: 60)phoA-DRcggtctagagcgccctgcaacgactgctgtt(SEQ ID NO: 61)PcrV1gaattcgaacaggaagaactgctg(SEQ ID NO: 62)PcrV2cggaagcttggatccaatggcactcagaatatca(SEQ ID NO: 63)HitA1ggatccggtggcggcggtagcg(SEQ ID NO: 64)HitA2aagcttttaatggtgatgatgatg(SEQ ID NO: 65)aUnderlining indicates restriction endonuclease recognition sequences.

TrimmingP. aeruginosato mitigate toxicity of outer membrane vesicles. A multitude of virulence factors produced by PA are involving in acute and chronic infections. Studies have illustrated that OMVs from WT PA can package numerous virulence factors, such as virulence effectors of the type III secretion system (T3SS) and toxins, and deliver them into host cells, impairing immune response and cytotoxicity. The toxins (ExoU, ExoT or ExoS) secreted by T3SS facilitated PA to breach in the epithelial barrier by antagonizing wound healing during colonization and promoting cell injury causing pneumonia. Also, several toxic effectors (Exotoxin A, LasA and LasB) of Type II secretion system (T2SS) contribute to bacterial pathogenicity. In addition, high levels of antibodies against alginate or elastases are induced upon PA infection, but these antibodies have poor opsonic activities, especially in CF individuals, fail to clear the infection effectively, and even exacerbate lung infection. Siderophores (pyochelin and pyoverdine), rhamnolipids, LPS, and alkaline phosphatases also facilitate PA infection. To mitigate toxicity caused by those factors, 14 genes (FIG.1A) were consecutively deleted to generate the PA-m14 mutant strain (Table 1). Mutations did not obviously alter morphology of PA-m14 in comparison to WT PA103 (FIG. S1), but the size of OMVs from PA-m14 was much smaller than that from WT PA103 (FIG.1B). Western blot showed that OMVs isolated from WT PA103 enclosed considerable amounts of the known toxins (ExoA and ExoU) (FIG.1C) that potentiate toxicity of OMVs to mammalian hosts. Following, in vivo toxicity testing of different OMVs showed that mice injected intramuscularly (i.m.) with 50 μg OMVs from WT PA103 succumbed within 3 days, but 80% mice survived by i.m. injection with 50 μg OMVs from PA-m1 strain with a single mutation of PA103_1714 (designated lpxL1) encoding lauroyltransferase that is response for the addition of laurate to lipid A. The PA103_1714 had 99.038% identity with PA3242 (designated HtrB2) in PAO1 strain. No mice succumbed by i.m. injection with 50 μg OMVs from PA-m6, PA-m11 or PA-m14, or even with 100 μg OMVs from PA-m14 (FIG.1D), implying that deletion of virulence factors and/or deduction of the fatty acid chain of lipid A significantly diminished the toxicity of PA OMVs. Regarding above results, the PA-m14 was chosen for the following studies.

Increasing PcrV-HitATfusion antigen enclosed byP. aeruginosaOMVs. Studies showed that active and passive immunization with the PcrV antigen or its protective antibody decreased lung inflammation and injury in a murine infection model and a burn mouse model. The components in PA iron acquisition systems, such as PA4359, PA4514, HitA (PA4687), and HitB (PA4688) are involved in iron transportation and associated with bacterial virulence, rendering them as potential vaccine candidates. Immunization with the ferric iron-binding periplasmic protein HitA provided protection for mice against systemic PA infection. PcrV and HitA antigen among different serotypes of PA has 98˜100% identity, respectively. However, OMVs directly isolated from PA-m14 strain contained low amounts of PcrV and HitA, which may limit OMV immunogenicity. In order to increase protective antigens enclosed in OMVs, PcrV (E28-I294, removing signal peptide) was fused with truncated HitA (D28-N355) from PA103 strain together designated as PcrV-HitAT (68 kDa, referred to PH) for the proof of concept. Therefore, the pSMV83 plasmid (Table 5 andFIG.24A) was constructed, in which the bla ss-pcrV-hitATis driven by strong Ptrcpromoter for high expression of the pcrV-hitATfusion gene in PA, and the bla ss encodes N-terminal β-lactamase signal peptide to facilitate secretion of the PH fusion antigen into periplasm of PA. Subsequently, the pSMV83 plasmid was introduced into PA-m14 strain to determine synthesis of PH antigen in bacteria and OMVs. Results showed that the PA-m14(pSMV83) strain synthesized significant amounts of PH and OMVs from this strain trapped decent amounts of PH antigen in comparison to PA-m14 harboring the empty plasmid pUCP20 (FIGS.24B& C)

Immunization with recombinant PA OMVs induces protection againstP. aeruginosainfection. Groups of mice (n=10, 5 males and 5 females) were intramuscularly (i.m.) immunized with 50 μg OMVs purified from PA-m14(pSMV83) (referred to OMV-PH) in 100 μl PBS, in which contains ˜10 μg PcrV-HitAT, and boosted at 21 days after prime immunization (FIG.25A). Immunization with 50 μg OMVs from PA-m14(pUCP20) (referred to OMV-NA), PH (10 μg)/alhydrogel or PBS/alhydrogel (PBS) as experimental controls. On day 42 after the initial vaccination, mice were challenged with PA by s.c. administration to mimic surgical infection or by i.n. route to mimic acute pneumonic infection. All OMV-immunized mice survived s.c. challenge with 7.4×107CFU (10 LD50) of PA103, while 40% of the PH-immunized mice survived the same challenge (FIG.25B). For i.n. challenge, vaccination with OMV-PH afforded 70% protection for mice infected with 6.5×106CFU (˜30 LD50) of PA103, but only 20% mice immunized with PH or OMV-NA survived the same challenge (FIG.25C). None of PBS-immunized mice survived both challenges (FIGS.25Band C).

Mice with OMV-PH immunization rapidly haltP. aeruginosainfection. On day 2 post infection, PBS-immunized mice had substantially higher PA titers (mean 7.2 log10CFU/g tissue) in lungs, spleens (mean 5.7 log10CFU/g tissue), livers (mean 5.6 log10CFU/g tissue) and bloods (mean 5.2 log10CFU/g tissue) than PH-, OMV-NA or OMV-PH immunized mice. In the PH-immunized mice, bacteria reached moderate levels in livers (mean 1.2 log10CFU/g tissue) and blood (mean 2.5 log10CFU/g tissue), but no bacteria were detected in spleens (FIG.25D). In the OMV-NA-immunized mice, bacteria reached moderate levels in spleens (mean 4.3 log10CFU/g tissue) and livers (mean 1.2 log10CFU/g tissue), but no bacteria were detected in blood (FIG.25D). No PA were disseminated to spleens, livers, and blood in OMV-PH-immunized mice (FIG.25D).

Mice with OMV-PH immunization rapidly haltP. aeruginosainfection. At 48 h after s.c. challenge with PA103, significant high bacteria titers were detected in livers (mean 4.2 log10CFU/g tissue) and blood (mean 4.6 log10CFU/g tissue) of PBS-immunized mice, moderate bacterial titers were observed in spleens (mean 2.7 log10CFU/g tissue) and lungs (mean 2.8 log10CFU/g tissue) of PBS-immunized mice. PH immunization could significantly reduce bacterial titers in livers, spleens, lungs, and blood, but not completely clear PA from livers, spleens, and blood at 48 h post infection. No bacteria were detected in OMV-NA or OMV-PH-immunized groups in all of tissues at 48 h post infection (FIG.25E). Data of bacteria burdens in mice organs after PA103 challenge could well correlate with animal survivals and host responses