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Antigen processing in the class II MHC pathway depends on conventional proteolytic enzymes, potentially acting on antigens in native-like conformational states. CD4+ epitope dominance arises from a competition between antigen folding, proteolysis, and MHCII binding. Protease-sensitive sites, linear antibody epitopes, and CD4+ T-cell epitopes were mapped in the plague vaccine candidate F1-V to evaluate the various contributions to CD4+ epitope dominance. Using X-ray crystal structures, antigen processing likelihood (APL) predicts CD4+ epitopes with significant accuracy without considering peptide-MHCII binding affinity. The profiles of conformational flexibility derived from the X-ray crystal structures of the F1-V proteins, Caf1 and LcrV, were similar to the biochemical profiles of linear antibody epitope reactivity and protease-sensitivity, suggesting that the role of structure in proteolysis was captured by the analysis of the crystal structures. The patterns of CD4+ T-cell epitope dominance in C57BL/6, CBA, and BALB/c mice were compared to epitope predictions based on APL, peptide binding to MHCII proteins, or both. For a sample of 13 diverse antigens larger than 200 residues, accuracy of epitope prediction by the combination of APL and I-Ab-MHCII-peptide affinity approached 40%. When MHCII allele specificity is also diverse, such as in human immunity, prediction of dominant epitopes by APL alone approached 40%. Since dominant CD4+ epitopes tend to occur in conformationally stable antigen domains, crystal structures typically are available for analysis by APL; and thus, the requirement for a crystal structure is not a severe limitation.

Mice immunized with a combination of an adenovirus vector (Ad5-YFV) and live-attenuated (LMA)-based vaccines were evaluated for protective efficacy against pneumonic plague. While the Ad5-YFV vaccine harbors a fusion cassette of three genes encodingYscF,F1, andLcrV, LMA represents a mutant of parentalYersinia pestisCO92 deleted for genes encodingLpp,MsbB, andAil. Ad5-YFV and LMA were either administered simultaneously (1-dose regimen) or 21 days apart in various order and route of administration combinations (2-dose regimen). The 2-dose regimen induced robust immune responses to provide full protection to animals against parental CO92 and its isogenic F1 (CAF-)-deletion mutant challenges during both short- and long-term studies. Mice intranasally (i.n.) immunized with Ad5-YFV first followed by LMA (i.n. or intramuscularly [i.m.]) had higher T- and B-cell proliferative responses and LcrV antibody titers than those in mice vaccinated with LMA (i.n. or i.m.) first ahead of Ad5-YFV (i.n.) during the long-term study. Specifically, the needle- and adjuvant-free vaccine combination (i.n.) is ideal for use in plague endemic regions. Conversely, with a 1-dose regimen, mice vaccinated with Ad5-YFV i.n. and LMA by the i.m. route provided complete protection to animals against CO92 and its CAF-mutant challenges, and elicited Th1/Th2, as well as Th17 responses, thus suitable for emergency vaccination during a plague outbreak or bioterrorist attack. This is a first study in which a viral vector-based and live-attenuated vaccines were effectively used in combination representing adjuvant- and/or needle- free immunization, with each vaccine triggering a distinct cellular immune response.ImportanceYersinia pestis, the causative agent of plague, is a Tier-1 select agent and a re-emerging human pathogen. A 2017 outbreak in Madagascar with >75% of cases being pneumonic and 8.6% causalities emphasized the importance of the disease. The World Health Organization has indicated an urgent need to develop new generation subunit and live-attenuated plague vaccines. We have developed a subunit vaccine including three components (YscF,F1, and LcrV) using an adenovirus platform (Ad5-YFV). In addition, we have deleted virulence genes ofY. pestis(e.g., lpp,msbB, andail) to develop a live-attenuated vaccine (LMA). Both of these vaccines generated robust humoral and cellular immunity and were highly efficacious in several animal models. We hypothesized the use of a heterologous prime-boost strategy or administrating both vaccines simultaneously could provide an adjuvant- and/or a needle- free vaccine(s) which have attributes of both vaccines for use in endemic regions and during an emergency situation.

Background Many pathogens, including Yersinia pestis, cannot be consistently and reliably cultured from blood. New approaches are needed to facilitate the detection of proteins, nucleic acid and microorganisms in whole blood samples to improve downstream assay performance. Detection of biomarkers in whole blood is difficult due to the presence of host proteins that obscure standard detection mechanisms. Nanotrap® particles are micron-sized hydrogel structures containing a dye molecule as the affinity bait and used to detect host biomarkers, viral nucleic acids and proteins as well as some bacterial markers. Nanotraps have been shown to bind and enrich a wide variety of biomarkers and viruses in clinically relevant matrices such as urine and plasma. Our objective was to characterize the binding ability of Nanotrap particle type CN3080 to Y. pestis bacteria, bacterial proteins and nucleic acids from whole human blood in order to potentially improve detection and diagnosis. Results CN3080 Nanotraps bind tightly to Yersinia bacteria, even after washing, and we were able to visualize the co-localized Nanotraps and bacteria by electron microscopy. These magnetic hydrogel Nanotraps were able to bind Yersinia DNA, supporting the utility of Nanotraps for enhancing nucleic acid-based detection methods. Nanotraps were capable of increasing Y. pestis nucleic acid yield by fourfold from whole human blood compared to standard nucleic acid extraction. Interestingly, we found CN3080 Nanotraps to have a high affinity for multiple components of the Yersinia type III secretion system (T3SS), including chaperone proteins, Yop effector proteins and virulence factor protein LcrV (V). Using Nanotraps as a rapid upstream sample-prep tool, we were able to detect LcrV in human blood by western blotting with minimal blood interference in contrast to direct western blotting of blood samples in which LcrV was obscured. We were able to computationally model the interaction of LcrV with the CN3080 Nanotrap dye and found that it had a low delta-G, suggesting high affinity. Importantly, Nanotraps were also able to enhance detection of secreted Yersinia proteins by mass spectrometry. Conclusion Upstream use of magnetic CN3080 Nanotrap particles may improve the downstream workflow though binding and enrichment of biomarkers and speed of processing. Utilization of Nanotrap particles can improve detection of Yersinia pestis proteins and nucleic acid from whole human blood and contribute to downstream assays and diagnostics including molecular methods such as sequencing and PCR and protein-based methods. Graphic Abstract

The causative agent of plague, Yersinia pestis, uses a type III secretion system to selectively destroy immune cells in humans, thus enabling Y. pestis to reproduce in the bloodstream and be transmitted to new hosts through fleabites. The host factors that are responsible for the selective destruction of immune cells by plague bacteria are unknown. Here we show that LcrV, the needle cap protein of the Y. pestis type III secretion system, binds to the N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Plague infection in mice is characterized by high mortality; however, Fpr1-deficient mice have increased survival and antibody responses that are protective against plague. We identified FPR1R190W as a candidate resistance allele in humans that protects neutrophils from destruction by the Y. pestis type III secretion system. Thus, FPR1 is a plague receptor on immune cells in both humans and mice, and its absence or mutation provides protection against Y. pestis. Furthermore, plague selection of FPR1 alleles appears to have shaped human immune responses towards other infectious diseases and malignant neoplasms. The receptor FPR1 on human immune cells interacts with Yersinia pestis, mutations in this receptor provide resistance against plague in humans and Fpr1 deficiency enhances survival in mice.

Yersinia enterocolitica causes a severe enteric infection in infants and young children. There is no vaccine approved for use in humans. We investigated the immunogenicity and protective capacity of Yersinia YopB, a conserved type III secretion system protein, alone or combined with LcrV in adult mice immunized intranasally. YopB or LcrV (5 μg) administered with the Escherichia coli double mutant heat-labile toxin (dmLT) adjuvant afforded modest (10–30%) protection against lethal Y. enterocolitica oral infection. The combination of YopB and LcrV (5 μg each) dramatically improved vaccine efficacy (70–80%). Additionally, it afforded complete protection against Y. pestis pulmonary infection. Immunization with YopB/LcrV+dmLT resulted in Ag-specific serum IgG, systemic and mucosal Ab-secreting cells, as well as IFN-γ, TNF-α, IL-2, IL-6, IL-17A, and KC production by spleen cells. Serum Abs elicited by YopB/LcrV+dmLT had enhanced bactericidal and opsonophagocytic killing activity. After Y. enterocolitica challenge, YopB/LcrV+dmLT–vaccinated mice exhibited intact intestinal tissue, active germinal centers in mesenteric lymph nodes, IgG+ and IgA+ plasmablasts in the lamina propria, and Abs in intestinal fluid. On the contrary, complete tissue destruction and abscesses were seen in placebo recipients that succumbed to infection. Mice immunized as infants with YopB+dmLT or LcrV+dmLT achieved 60% protection against lethal Y. enterocolitica infection, and vaccine efficacy increased to 90–100% when they received YopB/LcrV+dmLT. YopB+dmLT also afforded substantial (60%) protection when administered intradermally to infant mice. YopB/LcrV+dmLT is a promising subunit vaccine candidate with the potential to elicit broad protection against Yersinia spp.

International audience; Immunization with the live-attenuated Yersinia pseudotuberculosis VTnF1 strain producing a Yersinia pestis F1 pseudocapsule efficiently protects mice against bubonic and pneumonic plague. In clinical trials, demonstration of a plague vaccine's efficacy in humans will not be feasible, and correlates of protection will be needed to bridge the immune response of protected animals to that of vaccinated humans. Using serum transfer and vaccination of antibody-deficient µMT mice, we established that both humoral and cellular responses elicited by VTnF1 independently conferred protection against bubonic plague. Thus, correlates were searched for in both responses, using blood only. Mice were vaccinated with increasing doses of VTnF1 to provide a range of immune responses and survival outcomes. The cellular response was evaluated using an in vitro IFNγ release assay, and IFNγ levels were significantly associated with protection, although some survivors were negative for IFNγ, so that IFNγ release is not a fully satisfactory correlate. Abundant serum IgG against the F1 capsule, Yop injectable toxins, and also non-F1 Y.pestis antigens were found, but none against the LcrV antigen. All readouts correlated to survival and to each other, confirming that vaccination triggered multiple protective mechanisms developing in parallel. Anti-F1 IgG was the most stringent correlate of protection, in both inbred BALB/c mice and outbred OF1 mice. This indicates that antibodies (Ab) to F1 play a dominant role for protection even in the presence of Ab to many other targets. Easy to measure, the anti-F1 IgG titer will be useful to evaluate the immune response in other animal species and in clinical trials.

A plague vaccine with a fusion cassette of YscF, F1, and LcrV encoding genes in an adenovirus-5 vector (rAd5-YFV) is evaluated for efficacy and immune responses in mice. Two doses of the vaccine provides 100% protection when administered intranasally against challenge with Yersinia pestis CO92 or its isogenic F1 mutant in short- or long- term immunization in pneumonic/bubonic plague models. The corresponding protection rates drop in rAd5-LcrV monovalent vaccinated mice in plague models. The rAd5-YFV vaccine induces superior humoral, mucosal and cell-mediated immunity, with clearance of the pathogen. Immunization of mice with rAd5-YFV followed by CO92 infection dampens proinflammatory cytokines and neutrophil chemoattractant production, while increasing Th1- and Th2-cytokine responses as well as macrophage/monocyte chemo-attractants when compared to the challenge control animals. This is a first study showing complete protection of mice from pneumonic/bubonic plague with a viral vector-based vaccine without the use of needles and the adjuvant.

The recent progress in immunoinformatics provided the basis for an accelerated development of target-specific peptide vaccines as an alternative to the traditional vaccine concept. However, there is still limited information on whether the in silico predicted immunoreactive epitopes correspond to those obtained from the actual experiments. Here, humoral and cellular immune responses to two major Yersinia pestis protective antigens, F1 and LcrV, were studied in human donors immunized with the live plague vaccine (LPV) based on the attenuated Y. pestis strain EV line NIIEG. The F1 antigen provided modest specific cellular (mixed T helper 1 (Th1)/Th2 type) and humoral immune responses in vaccinees irrespective of the amount of annual vaccinations and duration of the post-vaccination period. The probing of the F1 overlapping peptide library with the F1-positive sera revealed the presence of seven linear B cell epitopes, which were all also predicted by in silico assay. The immunoinformatics study evaluated their antigenicity, toxicity, and allergenic properties. The epitope TSQDGNNH was mostly recognized by the sera from recently vaccinated donors rather than antibodies from those immunized decades ago, suggesting the usefulness of this peptide for differentiation between recent and long-term vaccinations. The in silico analysis predicted nine linear LcrV-specific B-cell epitopes, however, weak antibody and cellular immune responses prevented their experimental evaluation, indicating that LcrV is a poor marker of successful vaccination. No specific Th17 immune response to either F1 or LcrV was detected, and there were no detectable serum levels of F1-specific immunoglobulin A (IgA) in vaccinees. Overall, the general approach validated in the LPV model could be valuable for the rational design of vaccines against other neglected and novel emerging infections with high pandemic potency.

CRISPR-associated proteins that produce a signal in the presence of a target nucleic acid represent potentially powerful tools for diagnostics, but they also exhibit shortfalls that plague many CRISPR systems. For instance, not all targets elicit robust activity, which challenges the timely development of sensitive assays, and though many such tests have been reported, they often avoid discussion of the crRNA design and screening process. Here, motivated by the desire to detect the Yersinia pestis lcrV virulence gene, we detail the process involved in developing components for a CRISPR-based test that provides sensitive and specific identification of this sequence using Cas13a. This includes detailing the diversity of crRNA performance, identifying sequence that enable detection with attomolar sensitivity and species-level specificity, and presenting a method for simple streamlining of the crRNA screening process to allow for the high-throughput testing required for developing assay design rules in the future.

To counteract the deadly pathogens, i.e., Y. pestis, Y. enetrocolitica, and Y. pseudotuberculosis, we prepared a recombinant DNA construct lcrV-hsp70 encoding the bivalent fusion protein LcrV-HSP70. The lcrV gene of Y. pestis and hsp70 domain II DNA fragment of M. tuberculosis were amplified by PCR. The lcrV amplicon was first ligated in the pET vector using NcoI and BamHI restriction sites. Just downstream to the lcrV gene, the hsp70 domain II was ligated using BamHI and Hind III restriction sites. The in-frame and the orientation of cloned lcrV-hsp70 were checked by restriction analysis and nucleotide sequencing. The recombinant bivalent fusion protein LcrV-HSP70 was expressed in E. coli and purified by affinity chromatography. The vaccine potential of LcrV-HSP70 fusion protein was evaluated in formulation with alum. BALB/c mice were vaccinated, and the humoral and cellular immune responses were studied. The fusion protein LcrV-HSP70 induced a strong and significant humoral immune response in comparison to control animals. We also observed a significant difference in the expression levels of IFN-γ and TNF-α in LcrV–HSP70-immunized mice in comparison to control, HSP70, and LcrV groups. To test the protective efficacy of the LcrV–HSP70 fusion protein against plague and Yersiniosis, the vaccinated mice were challenged with Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis separately. The bivalent fusion protein LcrV–HSP70 imparted 100% protection against the plague. In the case of Yersiniosis, on day 2 post challenge, there was a significant reduction in the number of CFU of Y. enterocolitica and Y. pseudotuberculosis in the blood (CFU/ml) and the spleen (CFU/g) of vaccinated animals in comparison to the LcrV, HSP70, and control group animals.

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 × 105 CFU (80,000 50% lethal dose [LD50]) and intranasal challenge with 5 × 103 CFU (50 LD50) of virulent Y. pestis. This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

We incorporated the ΔP(fur)::TT araC P(BAD) fur deletion-insertion mutation on top of a previous Yersinia pseudotuberculosis mutant (Δasd ΔyopJ ΔyopK) to construct a new mutant designated as Yptb5, which manifests the arabinose-dependent regulated delayed fur (encoding ferric uptake regulator) shut-off. The Yptb5 strain was used to deliver an adjuvanted fusion protein, FliC180-LcrV. Levels of FliC180-LcrV synthesis were same in Yptb5 either harboring pSMV4, a p15A ori plasmid or pSMV8, a pSC101 ori plasmid containing the fliC180-lcrV fusion gene driven by P(trc) promoter. Tissue burdens of both Yptb5(pSMV4) and Yptb5(pSMV8) in mice had similar patterns. Mice vaccinated orally with 5×10(8) CFU of either Yptb5(pSMV4) or Yptb5(pSMV8) strain were primed high antibody titers with a balanced Th1/Th2 response, also developed potent T-cell responses with significant productions of IFN-γ, IL-17A and TNF-α. Immunization with each mutant strain conferred complete protection against pulmonary challenge with 5.5 × 10(3) CFU (55 LD(50)) of Y. pestis, but partial protection (50% survival) against 100 LD(50) of Y. pestis. Our results demonstrate that arabinose-dependent regulated delayed fur shut-off is an effective strategy to develop live attenuated bacterial vaccines while retaining strong immunogenicity.

The type III secretion system is a highly conserved virulence mechanism that is widely distributed in Gram-negative bacteria. It has a syringe-like structure composed of a multi-ring basal body that spans the bacterial envelope and a projecting needle that delivers virulence effectors into host cells. Here, we showed that the Yersinia inner rod protein YscI directly interacts with the needle protein YscF inside the bacterial cells and that this interaction depends on amino acid residues 83-102 in the carboxyl terminus of YscI. Alanine substitution of Trp-85 or Ser-86 abrogated the binding of YscI to YscF as well as needle assembly and the secretion of effectors (Yops) and the needle tip protein LcrV. However, yscI null mutants that were trans-complemented with YscI mutants that bind YscF still assembled the needle and secreted Yops, demonstrating that a direct interaction between YscF and YscI is critical for these processes. Consistently, YscI mutants that did not bind YscF resulted in greatly decreased HeLa cell cytotoxicity. Together, these results show that YscI participates in needle assembly by directly interacting with YscF.

Yersinia pestis, one of history's deadliest pathogens, has killed millions over the course of human history. It has attributes that make it an ideal choice to produce mass casualties and is a prime candidate for use as a biological weapon. When aerosolized, Y. pestis causes pneumonic plague, a pneumonia that is 100% lethal if not promptly treated with effective antibiotics. Currently, there is no FDA approved plague vaccine. The current lead vaccine candidate, a parenterally administered protein subunit vaccine comprised of the Y. pestis virulence factors, F1 and LcrV, demonstrated variable levels of protection in primate pneumonic plague models. As the most likely mode of exposure in biological attack with Y. pestis is by aerosol, this raises a question of whether this parenteral vaccine will adequately protect humans against pneumonic plague. In the present study we evaluated two distinct mucosal delivery platforms for the intranasal (IN) administration of LcrV and F1 vaccine proteins, a live bacterial vector, Lactobacillus plantarum, and a Tobacco Mosaic Virus (TMV) based delivery platform. IN administration of L. plantarum expressing LcrV, or TMV-conjugated to LcrV and F1 (TMV-LcrV+TMV-F1) resulted in the similar induction of high titers of IgG antibodies and evidence of proinflammatory cytokine secretion. However, only the TMV-conjugate delivery platform protected against subsequent lethal challenge with Y. pestis. TMV-LcrV+TMV-F1 co-vaccinated mice had no discernable morbidity and no mortality, while mice vaccinated with L. plantarum expressing LcrV or rLcrV+rF1 without TMV succumbed to infection or were only partially protected. Thus, TMV is a suitable mucosal delivery platform for an F1-LcrV subunit vaccine that induces complete protection against pneumonic infection with a lethal dose of Y. pestis in mice.

In this study, a novel recombinant attenuated Yersinia pseudotuberculosis PB1+ strain (χ10069) engineered with ΔyopK ΔyopJ Δasd triple mutations was used to deliver a Y. pestis fusion protein, YopE amino acid 1 to 138-LcrV (YopENt138-LcrV), to Swiss Webster mice as a protective antigen against infections by yersiniae. χ10069 bacteria harboring the pYA5199 plasmid constitutively synthesized the YopENt138-LcrV fusion protein and secreted it via the type 3 secretion system (T3SS) at 37°C under calcium-deprived conditions. The attenuated strain χ10069(pYA5199) was manifested by the establishment of controlled infection in different tissues without developing conspicuous signs of disease in histopathological analysis of microtome sections. A single-dose oral immunization of χ10069(pYA5199) induced strong serum antibody titers (log10 mean value, 4.2), secretory IgA in bronchoalveolar lavage (BAL) fluid from immunized mice, and Yersinia-specific CD4+ and CD8+ T cells producing high levels of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin 2 (IL-2), as well as IL-17, in both lungs and spleens of immunized mice, conferring comprehensive Th1- and Th2-mediated immune responses and protection against bubonic and pneumonic plague challenges, with 80% and 90% survival, respectively. Mice immunized with χ10069(pYA5199) also exhibited complete protection against lethal oral infections by Yersinia enterocolitica WA and Y. pseudotuberculosis PB1+. These findings indicated that χ10069(pYA5199) as an oral vaccine induces protective immunity to prevent bubonic and pneumonic plague, as well as yersiniosis, in mice and would be a promising oral vaccine candidate for protection against plague and yersiniosis for human and veterinary applications.

The mechanisms underlying the effects of γ-globulin therapy for bacterial infections are thought to involve bacterial cell lysis via complement activation, phagocytosis via bacterial opsonization, toxin neutralization, and antibody-dependent cell-mediated cytotoxicity. Nevertheless, recent advances in the study of pathogenicity in gram-negative bacteria have raised the possibility of an association between γ-globulin and bacterial toxin secretion. Over time, new toxin secretion systems like the type III secretion system have been discovered in many pathogenic gram-negative bacteria. With this system, the bacterial toxins are directly injected into the cytoplasm of the target cell through a special secretory apparatus without any exposure to the extracellular environment and, therefore, with no opportunity for antibodies to neutralize the toxin. However, because antibodies against the V-antigen, which is located on the needle-shaped tip of the bacterial secretion apparatus, can inhibit toxin translocation, this raises the hope that the toxin might be susceptible to antibody targeting. Because multi-drug resistant bacteria are now prevalent, inhibiting this secretion mechanism is attractive as an alternative or adjunctive therapy against lethal bacterial infections. Thus, it would not be unreasonable to define the blocking effect of anti-V-antigen antibodies as the fifth mechanism for immunoglobulin action against bacterial infections.

The V-antigen, a virulence-associated protein, was first identified inYersinia pestismore than half a century ago. Since then, other V-antigen homologs and orthologs have been discovered and are now considered vital molecules for the toxic effects mediated by the type III secretion system in infections caused by various pathogenic Gram-negative bacteria. After purifying recombinant V-antigen proteins including PcrV fromPseudomonas aeruginosa, LcrV fromYersinia, LssV fromPhotorhabdus luminescens, AcrV fromAeromonas salmonicida, and VcrV fromVibrio parahaemolyticus, we developed an enzyme-linked immunoabsorbent assay to measure titers against each V-antigen in the sera collected from 186 adult volunteers. Different titer-specific correlation levels were determined for the five V-antigens. The anti-LcrV and anti-AcrV titers shared the highest correlation with each other, with a correlation coefficient of 0.84. The next highest correlation coefficient was between anti-AcrV and anti-VcrV titers at 0.79, while the lowest correlation was found between anti-LcrV and anti-VcrV titers, which were still higher than 0.7 Sera from mice immunized with one of the five recombinant V-antigens displayed cross-antigenicity with some of the other four V-antigens, supporting the results from the human sera. Thus, the serum anti-V-antigen titer measurement system could potentially be used for epidemiological investigations on various pathogenic Gram-negative bacteria.

Plague caused by the Gram-negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non-human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V-OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV-based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo-tolerance; needle-free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell-mediated immune responses; and targeting of primary sites of plague infection.

Yersinioses caused by Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are significant concerns in human and veterinary health. The link between virulence and the potent LcrV antigen has prompted the latter's selection as a major component of anti-Yersinia vaccines. Here, we report that (i) the group of Yersinia species encompassing Y. pestis and Y. pseudotuberculosis produces at least five different clades of LcrV and (ii) vaccination of mice with an LcrV-secreting Lactococcus lactis only protected against Yersinia strains producing the same LcrV clade as that of used for vaccination. By vaccinating with engineered LcrVs and challenging mice with strains producing either type of LcrV or a LcrV mutated for regions of interest, we highlight key polymorphic residues responsible for the absence of cross-protection. Our results show that an anti-LcrV-based vaccine should contain multiple LcrV clades if protection against the widest possible array of Yersinia strains is sought.

Background Yersinia pestis, the etiological pathogen of plague, is capable of repressing the immune response of white blood cells to evade phagocytosis. The V-antigen (LcrV) was found to be involved in this process by binding to human Toll-like Receptor 2 (TLR2). The detailed mechanism behind this LcrV and TLR2 mediated immune response repression, however, is yet to be fully elucidated due to the lack of structural information. Results In this work, with protein structure modelling, we were able to construct a structure model of the heterotetramer of Y. pestis LcrV and human TLR2. Molecular dynamics simulation suggests the stability of this structure in aquatic environment. The LcrV model has a dumbbell-like structure with two globule domains (G1 at N-terminus and G2 away from membrane) connected with a coiled-coil linker (CCL) domain. The two horseshoe-shape TLR2 subunits form a V-shape structure, are not in direct contact with each other, and are held together by the LcrV homodimer. In this structure model, both the G1 and CCL domains are involved in the formation of LcrV homodimer, while all three domains are involved in LcrV-TLR2 binding. A mechanistic model was proposed based on this heterotetrameric structure model: The LcrV homodimer separates the TLR2 subunits to inhibit the dimerization of TLR2 and subsequent signal transfer for immune response; while LcrV could also inhibit the formation of heterodimers of TLR2 with other TLRs, and leads to immune response repression. Conclusions A heterotetrameric structure of Y. pestis LcrV and human TLR2 was modelled in this work. Analysis of this modelled structure showed its stability in aquatic environments and the role of LcrV domains and residues in protein-protein interaction. A mechanistic model for the role of LcrV in Y. pestis pathogenesis is raised based on this heterotetrameric structure model. This work provides a hypothesis of LcrV function, with which further experimental validation may elucidate the role of LcrV in human immune response repression.

Bacillus anthracis and Yersinia pestis are zoonotic bacteria capable of causing severe and sometimes fatal infections in animals and humans. Although considered as diseases of antiquity in industrialized countries due to animal and public health improvements, they remain endemic in vast regions of the world disproportionally affecting the poor. These pathogens also remain a serious threat if deployed in biological warfare. A single vaccine capable of stimulating rapid protection against both pathogens would be an extremely advantageous public health tool. We produced multiple-antigen fusion proteins (MaF1 and MaF2) containing protective regions from B. anthracis protective antigen (PA) and lethal factor (LF), and from Y. pestis V antigen (LcrV) and fraction 1 (F1) capsule. The MaF2 sequence was also expressed from a plasmid construct (pDNA-MaF2). Immunogenicity and protective efficacy were investigated in mice following homologous and heterologous prime-boost immunization. Antibody responses were determined by ELISA and anthrax toxin neutralization assay. Vaccine efficacy was determined against lethal challenge with either anthrax toxin or Y. pestis. Both constructs elicited LcrV and LF-specific serum IgG, and MaF2 elicited toxin-neutralizing antibodies. Immunizations with MaF2 conferred 100% and 88% protection against Y. pestis and anthrax toxin, respectively. In contrast, pDNA-MaF2 conferred only 63% protection against Y. pestis and no protection against anthrax toxin challenge. pDNA-MaF2-prime MaF2-boost induced 75% protection against Y. pestis and 25% protection against anthrax toxin. Protection was increased by the molecular adjuvant CARDif. In conclusion, MaF2 is a promising multi-antigen vaccine candidate against anthrax and plague that warrants further investigation., Author summary Anthrax and plague are ancient infectious diseases that continue to affect people living in poor, endemic regions and to threaten industrialized nations due to their potential use in biowarfare. Candidate vaccines need improvement to minimize non-desirable effects and increase their efficacy. The purpose of this work was to develop and evaluate a single subunit vaccine capable of conferring protection against Bacillus anthracis and Yersinia pestis. To this end, specific regions from their genome or key protective protein sequences from both microorganisms were combined to obtain either recombinant plasmids or recombinant proteins and tested as vaccine candidates in mice. The recombinant protein MaF2 induced specific antibody responses and afforded full and partial protection against Y. pestis and B. anthracis, respectively. Meanwhile, the DNA vaccine equivalent to MaF2 conferred only partial protection against Y. pestis, which increased when combined with an MaF2 protein boost. MaF2 emerged as a promising dual pathogen recombinant vaccine that warrants further investigation.

The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective protein that is considered as a vaccine component for humans. LcrV mediates the delivery of Yop toxins into host cells and upregulates TLR2-dependent IL-10 production. Although LcrV can interact with the receptor-bound human interferon-γ (hIFN-γ), the significance of these interactions in plague pathogenesis is not known. In this study, we determined the parameters of specific interactions of LcrV and LcrV68-326 with primary human thymocytes and Jurkat T-leukemia cells in the presence of receptor-bound hIFN-γ. Although the C-terminal region of hIFN-γ contains a GRRA138-141 site needed for high-affinity binding of LcrV and LcrV68-326, in the hIFN-γ homodimer, these GRRA138-141 target sites becomes accessible for targeting by LcrV or LcrV68-326 only after immobilization of the hIFN-γ homodimer on the hIFN-γ receptors of thymocytes or Jurkat T-cells. The interaction of LcrV or LcrV68-326 with receptor-bound hIFN-γ on the thymocytes or Jurkat T-cells caused apoptosis of both cell types, which can be completely blocked by the addition of monoclonal antibodies specific to the LEEL32-35 and DEEI203-206 sites of LcrV. The ability of LcrV to utilize hIFN-γ is insidious and may account in part for the severe symptoms of plague in humans.

Mouse monoclonal antibodies were raised against plague disease biomarkers: the bacterial capsular protein fraction 1 (F1) and the low-calcium response—LcrV virulence factor (Vag). A novel tandem assay, employing BioLayer Interferometry (BLI), enabled the isolation of antibodies against four different epitopes on Vag. The tandem assay was carried out with hybridoma supernatants, circumventing the need for antibody purification. The BioLayer assay was further adopted for characterization of epitope-repetitive antigens, enabling the discovery of two unique epitopes on F1. The selected antibodies were purified and applied as “oligo-clonal” reagents for the immuno-detection of both biomarkers. The developed Homogenous Time Resolved Fluorescence (HTRF) tests were short (10 min) and simple (no washing steps), allowing for detection of 10 ng/mL F1 and 2.5 ng/mL Vag. The tests were successfully applied for detection of disease biomarkers produced by various Y. pestis strains during growth in blood culture vials.

Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis . We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF , caf1 , and lcrV ). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models.

Multiplexed detection technologies are becoming increasingly important given the possibility of bioterrorism attacks, for which the range of suspected pathogens can vary considerably. In this work, we describe the use of Luminex MagPlex magnetic microspheres for the construction of two multiplexed diagnostic suspension arrays, enabling antibody-based detection of bacterial pathogens and their related disease biomarkers directly from blood cultures. The first 4-plex diagnostic array enabled the detection of both anthrax and plague infections using soluble disease biomarkers, including protective antigen (PA) and anthrax capsular antigen for anthrax detection and the capsular F1 and LcrV antigens for plague detection. The limits of detection (LODs) ranged between 0.5 and 5 ng/ml for the different antigens. The second 2-plex diagnostic array facilitated the detection of Yersinia pestis (LOD of 1 × 10 6 CFU/ml) and Francisella tularensis (LOD of 1 × 10 4 CFU/ml) from blood cultures. Inoculated, propagated blood cultures were processed (15 to 20 min) via 2 possible methodologies (Vacutainer or a simple centrifugation step), allowing the direct detection of bacteria in each sample, and the entire assay could be performed in 90 min. While detection of bacteria and soluble markers from blood cultures using PCR Luminex suspension arrays has been widely described, to our knowledge, this study is the first to demonstrate the utility of the Luminex system for the immunodetection of both bacteria and soluble markers directly from blood cultures. Targeting both the bacterial pathogens as well as two different disease biomarkers for each infection, we demonstrated the benefit of the multiplexed developed assays for enhanced, reliable detection. The presented arrays could easily be expanded to include antibodies for the detection of other pathogens of interest in hospitals or labs, demonstrating the applicability of this technology for the accurate detection and confirmation of a wide range of potential select agents.

A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen–antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17–470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

Recent studies on T follicular helper (Tfh) cells have significantly advanced our understanding of T cell-dependent B cell responses. However, little is known about the early stage of Tfh cell commitment by dendritic cells (DCs), particularly by the conventional CD8α(+) and CD8α(-) DC subsets. We show that CD8α(-) DCs localized at the interfollicular zone play a pivotal role in the induction of antigen-specific Tfh cells by upregulating the expression of Icosl and Ox40l through the non-canonical NF-κB signaling pathway. Tfh cells induced by CD8α(-) DCs function as true B cell helpers, resulting in significantly increased humoral immune responses against various human pathogenic antigens, including Yersinia pestis LcrV, HIV Gag, and hepatitis B surface antigen. Our findings uncover a mechanistic role of CD8α(-) DCs in the initiation of Tfh cell differentiation and thereby provide a rationale for investigating CD8α(-) DCs in enhancing antigen-specific humoral immune responses for improving vaccines and therapeutics.

Bacterium-like particles (BLPs), derived from Lactococcus lactis , offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring (PA) domain. In this study, the tip proteins IpaD, SipD, and LcrV belonging to type III secretion systems of Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica , respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:424-432, 2015

Glutathionylation, the formation of reversible mixed disulfides between glutathione and protein cysteine residues, is a posttranslational modification previously observed for intracellular proteins of bacteria. Here we show that Yersinia pestis LcrV, a secreted protein capping the type III secretion machine, is glutathionylated at Cys273 and that this modification promotes association with host ribosomal protein S3 (RPS3), moderates Y. pestis type III effector transport and killing of macrophages, and enhances bubonic plague pathogenesis in mice and rats. Secreted LcrV was purified and analyzed by mass spectrometry to reveal glutathionylation, a modification that is abolished by the codon substitution Cys273Ala in lcrV. Moreover, the lcrVC273A mutation enhanced the survival of animals in models of bubonic plague. Investigating the molecular mechanism responsible for these virulence attributes, we identified macrophage RPS3 as a ligand of LcrV, an association that is perturbed by the Cys273Ala substitution. Furthermore, macrophages infected by the lcrVC273A variant displayed accelerated apoptotic death and diminished proinflammatory cytokine release. Deletion of gshB, which encodes glutathione synthetase of Y. pestis, resulted in undetectable levels of intracellular glutathione, and we used a Y. pestis ΔgshB mutant to characterize the biochemical pathway of LcrV glutathionylation, establishing that LcrV is modified after its transport to the type III needle via disulfide bond formation with extracellular oxidized glutathione., IMPORTANCE Yersinia pestis, the causative agent of plague, has killed large segments of the human population; however, the molecular bases for the extraordinary virulence attributes of this pathogen are not well understood. We show here that LcrV, the cap protein of bacterial type III secretion needles, is modified by host glutathione and that this modification contributes to the high virulence of Y. pestis in mouse and rat models for bubonic plague. These data suggest that Y. pestis exploits glutathione in host tissues to activate a virulence strategy, thereby accelerating plague pathogenesis.

The risk of plague as a bioweapon has prompted increasing research efforts to develop plague vaccines due to its extreme virulence and the ease of its transmission. Subunit vaccines that contain F1 and LcrV antigens of Y. pestis have been tested for safety and immunogenicity, but doubts have been raised about whether subunit vaccines that engender antibody responses will protect against pneumonic plague, which requires both humeral and cellular immune responses for protection. The live, attenuated vaccine EV76, a pgm locus deficient Y. pestis strain, has been used for a long time in the Former Soviet Union and some Asian countries, but is not commercially available in the US and Europe due to safety concerns. However, the live attenuated Y. pestis vaccines are still considered to be the most effective way to prevent plague. In this review, we present our opinions about rationally creating live, safe and immunogenic Y. pestis vaccines with potential use for human based on established researches.