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Yersinia pestis is a Gram-negative bacterium that is the causative agent of plague and is widely recognized as a potential biological weapon. Due to the high fatality rate of plague when diagnosis is delayed, the development of rapid, sensitive, specific, and cost-effective methods is needed for its diagnosis. The Y. pestis low calcium response V (LcrV) protein has been identified as a potential microbial biomarker for the diagnosis of plague. In this paper, we present a highly sensitive, paper-based, vertical flow immunoassay (VFI) prototype for the detection of LcrV and the diagnosis of plague. An antigen-capture assay using monoclonal antibodies is employed to capture and detect the LcrV protein, using a colorimetric approach. In addition, the effect of miniaturizing the VFI device is explored based on two different sizes of VFI platforms, denoted as "large VFI" and "mini VFI." Also, a comparative analysis is performed between the VFI platform and a lateral flow immunoassay (LFI) platform to exhibit the improved assay sensitivity suitable for point-of-care (POC) diagnostics. The analytical sensitivity or limit of detection (LOD) in the mini VFI is approximately 0.025 ng/mL, that is, 10 times better than that of the large VFI platform or 80 times over a standard lateral flow configuration. The low LOD of the LcrV VFI appears to be highly suitable for testing clinical samples and potentially diagnosing plague at earlier time points. In addition, optimization of the gold nanoparticle (AuNP) concentration, nanomaterial plasmonic properties, and flow velocity analysis could improve the performance of the VFI. Furthermore, we developed automated image analysis software that shows potential for integrating the diagnostic system into a smartphone. These methods and findings demonstrate that the VFI platform is a highly sensitive device for detecting the LcrV and potentially many other biomarkers.

The paper reports the investigation of the population structure of Yersinia pestis strains belonging to the ulegeica subspecies (genetic branch 0.PE5), which is endemic to the natural plague foci in Mongolia and which is characterized by low epidemic potential compared to the strains belonging to the main plague subspecies. Whole genome SNP analysis of 12 Y. pestis subspecies ulegeica strains isolated in Mongolia considering the 1581 identified core SNPs revealed two main genetic clusters in correspondence with the geographic distribution of these strains. The first cluster, named 0.PE5/1, contains the strains isolated in the Umnegov’, Uverkhangai, and Gov’-Altai aimaks located in the south of Mongolia. The second cluster 0.PE5/2 comprises two subclusters, namely, 0.PE5/2-1, containing the strains from the Bayan-Ulgii and Khovd aimaks, and 0.PE5/2-2, represented solely by the strains isolated in the Bayan-Ulgii aimak. To find the possible causes of the selective virulence of subspecies ulegeica strains, the analysis of the nucleotide sequences of 79 pathogenicity factors along with some other genomic regions has been carried out. Specific mutations have been found in the following genes: irp1, included in the high-pathogenicity island of the chromosomal pigmentation region, and yscQ, involved in the regulation of the synthesis of the LcrV protein and outer membrane proteins (Yops) associated with plague pathogen virulence.

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.

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.

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.

Yersinia pestis, the causative agent of plague, has killed millions throughout human history. Though public health initiatives have reduced the number of plague cases, it remains endemic in many areas of the world. It also remains a significant threat for use as a biological weapon. Naturally occurring multi-drug antibiotic resistance has been observed in Y. pestis, and resistant strains have been engineered for use as a biological weapon. Vaccines represent our best means of protection against the threat of antibiotic resistant plague. We have developed a vaccine consisting of two Y. pestis virulence factors, LcrV (V) and F1, conjugated to Tobacco Mosaic Virus (TMV), a safe, non-replicating plant virus that can be administered mucosally, providing complete protection against pneumonic plague, the deadliest form of the disease and the one most likely to be seen in a biological attack. A single intranasal (i.n.) dose of TMV-F1 + TMV-V (TMV-F1/V) protected 88% of mice against lethal challenge with 100 LD50 of Y. pestis CO92pgm-, while immunization with rF1 + rV without TMV was not protective. Serum and tissues were collected at various timepoints after challenge to assess bacterial clearance, histopathology, cytokine production, and antibody production. Overall, TMV-F1/V immunized mice showed a significant reduction in histopathology, bacterial burden, and inflammatory cytokine production following challenge compared to rF1 + rV vaccinated and unvaccinated mice. Pneumonic challenge resulted in systemic dissemination of the bacteria in all groups, but only TMV-F1/V immunized mice rapidly cleared bacteria from the spleen and liver. There was a direct correlation between pre-challenge serum F1 titers and recovery in all immunized mice, strongly suggesting a role for antibody in the neutralization and/or opsonization of Y. pestis in this model. Mucosal administration of a single dose of a Y. pestis TMV-based subunit vaccine, without any additional adjuvant, can effectively protect mice from lethal infection.

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.

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.

The mechanisms underlying the effects of immunoglobulins on 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 the pathogenicity of Gram-negative bacteria have raised the possibility of an association between immunoglobulin 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, antibodies against the V-antigen, which is located on the needle-shaped tip of the bacterial secretion apparatus, can inhibit toxin translocation, thus raising the hope that the toxin may be susceptible to antibody targeting. Because multi-drug resistant bacteria are now prevalent, inhibiting this secretion mechanism is an attractive alternative or adjunctive therapy against lethal bacterial infections. Thus, it is not unreasonable to define the blocking effect of anti-V-antigen antibodies as the fifth mechanism for immunoglobulin action against bacterial infections.

In the current antibiotic resistance scenario, vaccines may provide best defense against lethal bacterial diseases. So far, there is no ideal vaccine available against plague. Despite providing complete protection in small animal models, F1/LcrV based vaccine failed to provide ideal protection in non human primates. Here, we cloned, expressed and purified YopE of Yersinia pestis and flagellin C (FliC) of Salmonella Typhi. However the best possible protection needs the significant induction of IFN-γ and TNF-α. To determine the protective potential of the recombinant YopE alone or in formulation with FliC, Balb/C mice were immunized subcutaneously. The formulations were prepared with alum, a human compatible adjuvant. In our studies, the combination of YopE + FliC induced significantly strong humoral and cellular immune responses. A combination of YopE + FliC provided 83% protection whereas YopE alone provided only 50% against 100LD50 of Y. pestis in a mouse model.

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.

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.

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.

Plague is one of the world’s most lethal human diseases caused by Yersinia pestis, a Gram-negative bacterium. Despite overwhelming studies for many years worldwide, there is no safe and effective vaccine against this fatal disease. Inhalation of Y. pestis bacilli causes pneumonic plague, a fast growing and deadly dangerous disease. F1/LcrV-based vaccines failed to provide adequate protection in African green monkey model in spite of providing protection in mice and cynomolgus macaques. There is still no explanation for this inconsistent efficacy, and scientists leg behind to search reliable correlate assays for immune protection. These paucities are the main barriers to improve the effectiveness of plague vaccine. In the present scenario, one has to pay special attention to elicit strong cellular immune response in developing a next-generation vaccine against plague. Here, we review the scientific contributions and existing progress in developing subunit vaccines, the role of molecular adjuvants; DNA vaccines; live delivery platforms; and attenuated vaccines developed to counteract virulent strains of Y. pestis.

For about four decades, hybridoma technologies have been the “work horse” of monoclonal antibody production. These techniques proved to be robust and reliable, albeit laborious. Over the years, several major improvements have been introduced into the field, but yet, antibody production still requires many hours of labor and considerable resources. In this work, we present a leap forward in the advancement of hybridoma-based monoclonal antibody production, which saves labor and time and increases yield, by combining hybridoma technology, fluorescent particles and fluorescence-activated cell sorting (FACS). By taking advantage of the hybridomas’ cell-surface associated antibodies, we can differentiate between antigen-specific and non-specific cells, based on their ability to bind the particles. The speed and efficiency of antibody discovery, and subsequent cell cloning, are of high importance in the field of infectious diseases. Therefore, as a model system, we chose the protein LcrV, a major virulence factor of the plague pathogen Yersinia pestis, an important re-emerging pathogen and a possible bioterror agent.

Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1 − strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1 − mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1 − CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.

Subunit antigen-based vaccines can provide a number of important benefits over traditional vaccine candidates, such as overall safety. However, because of the inherently low immunogenicity of these antigens, methods for colocalized delivery of antigen and immunostimulatory molecules (i.e., adjuvants) are needed. Here we report a robust nanolipoprotein particle (NLP)-based vaccine delivery platform that facilitates the codelivery of both subunit antigens and adjuvants. Ni-chelating NLPs (NiNLPs) were assembled to incorporate the amphipathic adjuvants monophosphoryl lipid A and cholesterol-modified CpG oligodeoxynucleotides, which can bind His-tagged protein antigens. Colocalization of antigen and adjuvant delivery using the NiNLP platform resulted in elevated antibody production against His-tagged influenza hemagglutinin 5 and Yersinia pestis LcrV antigens. Antibody titers in mice immunized with the adjuvanted NLPs were 5-10 times higher than those observed with coadministration formulations and nonadjuvanted NiNLPs. Colocalized delivery of adjuvant and antigen provides significantly greater immune stimulation in mice than coadministered formulations.

Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1–lcrV and caf1–lcrV–hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1–lcrV and caf1–lcrV–hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1–LcrV and F1–LcrV–HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1–LcrV and F1–LcrV–HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it’s isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1–LcrV–HSP70(II) sera in comparison to anti-F1–LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1–LcrV–HSP(II) immunized mice in comparison to F1–LcrV. Both F1–LcrV and F1–LcrV–HSP70(II) provided 100% protection. Our research findings suggest that F1–LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model.

Based on our improved novel Salmonella vaccine delivery platform, we optimized the recombinant attenuated Salmonella typhimurium vaccine (RASV) χ12094 to deliver multiple Yersinia pestis antigens. These included LcrV196 (amino acids, 131-326), Psn encoded on pYA5383 and F1 encoded in the chromosome, their synthesis did not cause adverse effects on bacterial growth. Oral immunization with χ12094(pYA5383) simultaneously stimulated high antibody titers to LcrV, Psn and F1 in mice and presented complete protection against both subcutaneous (s.c.) and intranasal (i.n.) challenges with high lethal doses of Y. pestis CO92. Moreover, no deaths or other disease symptoms were observed in SCID mice orally immunized with χ12094(pYA5383) over a 60-day period. Therefore, the trivalent S. typhimurium-based live vaccine shows promise for a next-generation plague vaccine.

The recent development of a homogeneous time-resolved Forster resonance energy transfer (TR–FRET) immunoassay enables one-step, rapid (minutes), and direct detection compared to the multistep, time-consuming (hours), heterogeneous ELISA-type immunoassays. The use of the time-resolved effect of a donor lanthanide complex with a delay time of microseconds and large Stokes shift enables the separation of positive signals from the background autofluorescence of the sample. However, this study shows that the sample matrices directly interfere with donor fluorescence and that interference cannot be eliminated by time-resolved settings alone. Moreover, the reduction in donor emission did not appear to be equivalent to the reduction in acceptor emission, resulting in incorrect FRET signal measurements. To overcome this limitation, an internal standard approach was developed using an isotype control antibody. This new approach was used to develop TR–FRET assays for rapid detection (15–30 min) of Bacillus anthracis spores and botulinum toxin (type E) in beverages, which are major concerns in bioterrorism involving deliberate food contamination. Additionally, we demonstrate the detection of B. anthracis-secreted protective antigen (PA) and the Yersinia pestis-secreted markers F1 and LcrV in blood cultures, which are early markers of bacteremia in infected hosts following a possible bioterror attack. The use of an internal standard enables the calculation of correct ΔF values without the need for an external standard. Thus, the use of the internal standard approach in homogeneous immunoassays facilitates the examination of any sample regardless of its origin, and therefore expands the applicability of TR–FRET assays for complex matrices.

Plague caused by Yersinia pestis is an ancient disease, responsible for millions of deaths in human history. Unfortunately, there is no FDA-approved vaccine available. Recombinant subunit vaccines based on two major antigens, Caf 1 (F1) and LcrV (V), have been under investigation and showed promise. However, there are two main problems associated with these vaccines. First, the Yersinia capsular protein F1 has high propensity to aggregate, particularly when expressed in heterologous systems such as Escherichia coli, thus affecting vaccine quality and efficacy. Second, the subunit vaccines do not induce adequate cell-mediated immune responses that also appear to be essential for optimal protection against plague. We have developed two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that may overcome these problems. First, by engineering F1 protein, we generated a monomeric and soluble F1V mutant (F1mutV) which has similar immunogenicity as wild-type F1V. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to retain a key CD4(+) T cell epitope. Second, we generated a nanoparticle plague vaccine that can induce balanced antibody- and cell-mediated immune responses. This was done by arraying the F1mutV on phage T4 via the small outer capsid (Soc) protein which binds to T4 capsid at nanomolar affinity. Preparation of these vaccines is described in detail and we hope that these would be considered as candidates for licensing a next-generation plague vaccine.

Yersinia pestis is the causative agent of plague, an ancient scourge that precipitates intermittent pandemics [1, 2]. Plague is a disease of mammals that is transmitted by fleabite or aerosol droplets, causing either bubonic or pneumonic plague [3]. Inoculation of humans with live-attenuated Y. pestis strains, identified as nonpigmented (pgm) colonies on hemin agar, affords protection against bubonic and pneumonic plague [4]. Nonpigmented strains, most notably Y. pestis EV76, have been used extensively as live-attenuated vaccines to successfully stem the spread of the last plague pandemic in Asia [5]. The pathogenesis of Y. pestis infections relies on the pathogen's type III secretion pathway, which delivers Yop effectors into immune cells via type III needle complexes that are capped by the protective antigen LcrV [6–8]. The protective immunity raised by live-attenuated plague vaccines is based on antibodies that are directed against the fraction 1 capsular antigen (Caf1, or F1 pilus) of Y. pestis and that interfere with the delivery of effectors by LcrV-capped type III needle complexes [9, 10]. Y. pestis variants defective for the expression of F1 capsular antigen caused plague in animals that had been immunized with the live-attenuated vaccine, indicating that protection is not universal for all Y. pestis isolates [10]. Spontaneous excision and loss of the 102-kb high-pathogenicity island and pigmentation (pgm) locus occurs on the Y. pestis chromosome at flanking IS100 elements [11, 12]. Y. pestis pgm strains are attenuated in animal models of bubonic and pneumonic plague because of their inability to synthesize yersiniabactin, a siderophore that scavenges iron from transferrin during host infection [13, 14]. Live-attenuated pgm plague vaccine–related illnesses were not reported during the immunization campaign involving Asian populations [5]. However, in a small clinical trial in the United States, some recipients of the live-attenuated plague vaccine experienced malaise, fever, and severe reactogenicity requiring hospitalization [4]. Recently, the nonpigmented Y. pestis strain UC91309 was isolated from a researcher with fatal, septicemic plague [15]. Genome sequencing suggested that the isolated strain had been acquired in the laboratory [15]. Autopsy findings included abnormally high levels of iron deposits in the liver and markedly elevated levels of serum ferritin, iron, total iron-binding capacity, and iron saturation [15]. The presence of a HFE C282Y mutation supported the postmortem diagnosis of hereditary hemochromatosis [15]. These clinical and pathological findings suggested that iron overload in tissues of an individual with hereditary hemochromatosis may complement the iron-scavenging defect in mutants that are unable to synthesize yersiniabactin, thereby restoring the virulence of pgm variants and their ability to cause plague. Formal proof for this hypothesis is not yet available. To address this question, we analyzed the virulence properties of Y. pestis UC91309 and used a mouse model of hereditary hemochromatosis to examine whether increased iron load in host tissues may restore the virulence of nonpigmented Y. pestis strains. Further, we asked whether the rV10-2 plague subunit vaccine [16], which elicits LcrV-specific antibodies that block Y. pestis type III injection of immune cells [17], can protect mice with hereditary hemochromatosis against plague disease.

Yersinia pestis is the causative agent responsible for bubonic and pneumonic plague. The bacterium uses the pLcr plasmid-encoded type III secretion system to deliver virulence factors into host cells. Delivery requires ATP hydrolysis by the YscN ATPase encoded by the yscN gene also on pLcr. A yscN mutant was constructed in the fully virulent CO92 strain containing a nonpolar, in-frame internal deletion within the gene. We demonstrate that CO92 with a yscN mutation was not able to secrete the LcrV protein (V-Antigen) and attenuated in a subcutaneous model of plague demonstrating that the YscN ATPase was essential for virulence. However, if the yscN mutant was complemented with a functional yscN gene in trans, virulence was restored. To evaluate the mutant as a live vaccine, Swiss-Webster mice were vaccinated twice with the ΔyscN mutant at varying doses and were protected against bubonic plague in a dose-dependent manner. Antibodies to F1 capsule but not to LcrV were detected in sera from the vaccinated mice. These preliminary results suggest a proof-of-concept for an attenuated, genetically engineered, live vaccine effective against bubonic plague.

Live plague vaccines have saved thousands of human lives in the 20th century and have continued to be used in Russia and other countries of the former Soviet Union for prophylaxis of plague. A live attenuated Y. pestis EV strain line NIIEG is used for the routine annual vaccination of plague workers, as well as people from the groups at risk. This vaccination can offer immunity against both bubonic and pneumonic plague. However, serologic markers of the human response to vaccination with EV NIIEG are poorly investigated. It is not clear whether other antigens, in addition to the established capsular antigen F1 and lipopolysaccharide, can elicit specific and long-lasting antibody responses in vaccinees. In this study, a humoral immunity to a panel of recombinant Y. pestis-specific antigens, such as F1, Pla, LcrV, YopM and YscF, was examined in volunteers, who received multiple annual immunizations with EV NIIEG during the period of 5 to 30 years. To evaluate a long-term immune response to these antigens, we chose a cohort of donors, who had their last immunization 4-30 years ago. The immunoblotting technique revealed that sera of 14 out of 17 donors contained antibodies to at least one of the tested antigens. As expected, the occurrence of anti-F1 antibodies was detected in a large group of vaccinees (57%). In contrast, the presence of specific antibodies to either LcrV or YscF was less pronounced (26% and 36%, respectively), and only two donors possessed anti-YopM (10%). Surprisingly, we found that sera of the vast majority of volunteers (82%) gave positive reaction with the outer membrane protease Pla, specific to Y. pestis. This analysis will provide insights into the correlates of immunity elicited in humans by live plague vaccine, aid in the search for markers of exposure to plague, and help to develop new diagnostic protocols.

Yersinia pestis is the causative agent of the most deadly disease plague. F1 and V antigens are the major vaccine candidates. Six protective epitopes of V antigen of varying length (15-25aa) were assembled on a lysine backbone as multiple antigen peptide (MAP) using standard Fmoc chemistry. Palmitate was coupled at amino terminus end. Amino acid analysis, SDS-PAGE, immunoblot and immunoreactivity proved the authenticity of MAP. MAP was immunized intranasally encapsulated in PLGA (polylactide-co-glycolide) microspheres and with/without/adjuvants murabutide and CpG ODN 1826 (CpG), in three strains of mice. Humoral and mucosal immune responses were studied till day 120 and memory response was checked after immunization with native V antigen on day 120. Epitope specific serum and mucosal washes IgG, IgA, IgG subclasses and specific activity were measured by indirect ELISA and sandwich ELISA, respectively. IgG and IgA peak antibody titers of all the MAP construct formulations in sera were ranging from 71,944 to 360,578 and 4493 to 28,644, respectively. MAP with CpG showed significantly high (p0.0001) antibody titers ranging from 101,690 to 360,578 for IgG and 28,644 for IgA. Mucosal peak IgG and IgA titers were ranging from 1425 to 8072 and 1425 to 7183, respectively in intestinal washes and 799-4528 and 566-4027, respectively in lung washes. MAP with CpG showed significantly high (p0.001) SIgA titers of 8000 in lung and 16,000 in intestinal washes. IgG isotyping revealed IgG2a/IgG1 ratio1 with CpG. Serum and mucosal antipeptide IgG and IgA specific activities correlated well with antibody titers. All the constituent peptides contributed towards immune response. Structural analysis of MAP revealed little or no interaction between the peptides. Present study showed MAP to be highly immunogenic with high and long lasting antibody titers in serum and mucosal washes with good recall response with/without CpG as an adjuvant which can be used for vaccine development for plague.

We have shown that intranasal coapplication of Bacillus anthracis protective Ag (PA) together with a B. anthracis edema factor (EF) mutant having reduced adenylate cyclase activity (i.e., EF-S414N) enhances anti-PA Ab responses, but also acts as a mucosal adjuvant for coadministered unrelated Ags. To elucidate the role of edema toxin (EdTx) components in its adjuvanticity, we examined how a PA mutant lacking the ability to bind EF (PA-U7) or another mutant that allows the cellular uptake of EF, but fails to efficiently mediate its translocation into the cytosol (PA-dFF), would affect EdTx-induced adaptive immunity. Native EdTx promotes costimulatory molecule expression by macrophages and B lymphocytes, and a broad spectrum of cytokine responses by cervical lymph node cells in vitro. These effects were reduced or abrogated when cells were treated with EF plus PA-dFF, or PA-U7 instead of PA. We also intranasally immunized groups of mice with a recombinant fusion protein of Yersinia pestis F1 and LcrV Ags (F1-V) together with EdTx variants consisting of wild-type or mutants PA and EF. Analysis of serum and mucosal Ab responses against F1-V or EdTx components (i.e., PA and EF) revealed no adjuvant activity in mice that received PA-U7 instead of PA. In contrast, coimmunization with PA-dFF enhanced serum Ab responses. Finally, immunization with native PA and an EF mutant lacking adenylate cyclase activity (EF-K346R) failed to enhance Ab responses. In summary, a fully functional PA and a minimum of adenylate cyclase activity are needed for EdTx to act as a mucosal adjuvant.

Yersinia pestis, the causative organism of plague, is a zoonotic organism with a worldwide distribution. Although the last plague epidemic occurred in early 1900s, human cases continue to occur due to contact with infected wild animals. In this study, we have developed a reservoir-targeted vaccine against Y. pestis, to interrupt transmission of disease in wild animals as a potential strategy for decreasing human disease. A vaccinia virus delivery system was used to express the F1 capsular protein and the LcrV type III secretion component of Y. pestis as a fusion protein. Here we show that a single dose of this vaccine administered orally, generates a dose-dependent antibody response in mice. Antibody titers peak by 3 weeks after administration and remain elevated for a minimum of 45 weeks. Vaccination provided up to 100% protection against challenge with Y. pestis administered by intranasal challenge at 10 times the lethal dose with protection lasting a minimum of 45 weeks. An orally available, vaccinia virus expressed vaccine against Y. pestis may be a suitable vaccine for a reservoir targeted strategy for the prevention of enzootic plague.

Markers of the early stages of plague, a rapidly progressing deadly disease, are crucial for enabling the onset of an effective treatment. Here, we show that V-antigen protein (LcrV) is accumulated in the serum of Yersinia pestis-infected mice before bacterial colonization of the spleen and dissemination to blood, in a model of bubonic plague. LcrV accumulation is detected earlier than that of F1 capsular antigen, an established marker of disease. In a mouse model of pneumonic plague, LcrV can be determined in the bronchoalveolar lavage fluid somewhat later than F1, but before dissemination of Y. pestis to the blood. Thus, determination of soluble LcrV is suggested as a potential useful tool for monitoring disease progression in both bubonic and pneumonic plague. Moreover, it may be of particular advantage in cases of infections with F1 nonproducing strains.

The potential application of Yersinia pestis for bioterrorism emphasizes the urgent need to develop more effective vaccines against airborne infection. The current status of plague vaccines has been reviewed. The present emphasis is on subunit vaccines based on the F1 and LcrV antigens. These provide good protection in animal models but may not protect against F1 strains with modifications to the type III secretion system. The duration of protection against pneumonic infection is also uncertain. Other strategies under investigation include defined live-attenuated vaccines, DNA vaccines, mucosal delivery systems and heterologous immunization. The live-attenuated strain Y. pestis EV NIIEG protects against aerosol challenge in animal models and, with further modification to reduce residual virulence and to optimize respiratory protection, it could provide a shortcut to improved vaccines. The regulatory problems inherent in licensing vaccines for which efficacy data are unavailable and their possible solutions are discussed herein.

In previous studies, we demonstrated protection against plague in mice and prairie dogs using a raccoon pox (RCN) virus-vectored vaccine that expressed the F1 capsular antigen of Yersinia pestis . In order to improve vaccine efficacy, we have now constructed additional RCN-plague vaccines containing two different forms of the lcrV (V) gene, including full-length (Vfull) and a truncated form (V307). Mouse challenge studies with Y. pestis strain CO92 showed that vaccination with a combination of RCN-F1 and the truncated V construct (RCN-V307) provided the greatest improvement ( P = 0.01) in protection against plague over vaccination with RCN-F1 alone. This effect was mediated primarily by anti-F1 and anti-V antibodies and both contributed independently to increased survival of vaccinated mice.

Yersinia pestis is the causative agent of bubonic and pneumonic plague, human diseases with high mortality. Due to the microbe's ability to spread rapidly, plague epidemics present a serious public health threat. A search for prophylactic measures was initially based on historical reports of bubonic plague survivors and their apparent immunity. Due to safety and efficacy concerns, killed whole-cell preparations or live-attenuated plague vaccines are no longer considered in the United States. Vaccine developers have focused on specific subunits of plague bacteria. LcrV, a protein at the tip of type III secretion needles, and F1, the capsular pilus antigen, are both recognized as plague protective antigens. Antibodies against LcrV and F1 interfere with Y. pestis type III injection of host cells. While LcrV is absolutely essential for Y. pestis virulence, expression of F1 is dispensable for plague pathogenesis in small animals, non-human primates and presumably also in humans. Several subunit vaccines, for example rF1+rV (rYP002), rF1V or rV10, are being developed to generate plague protection in humans. Efficacy testing and licensure for human use requires the establishment of correlates for plague immunity.

Mab7.3 to Yersinia pestis LcrV antigen (LcrV(Ype)) protected J774A.1 macrophages in vitro from killing by a Yersinia pseudotuberculosis strain expressing LcrV(Ype). Of 4 site-directed mutations in the coiled-coil region (148-169) and 7 mutations in the 225-255 sequence of LcrV(Ype), only the mutation of N255 to D255, abrogated the binding of Mab7.3 and reduced its protective capacity against plague. Since the Mab7.3 epitope in LcrV(Ype) (135-275) encompasses a region (136-180) thought to be exposed on the injectisome, we suggest that Mab7.3 protects by binding to LcrV(Ype) and interfering with protein-protein interactions necessary for type three secretion.

Yersinia pestis , the agent of plague, uses a type III secretion injectisome to deliver Yop proteins into macrophages to counteract phagocytosis and induce apoptosis. Additionally, internalized Y. pestis can survive in the phagosomes of naïve or gamma interferon (IFN-γ)-activated macrophages by blocking vacuole acidification. The Y. pestis LcrV protein is a target of protective antibodies. The binding of antibodies to LcrV at the injectisome tip results in neutralization of the apoptosis of Y. pestis -infected macrophages and is used as an in vitro correlate of protective immunity. The cytokines IFN-γ and tumor necrosis factor alpha can cooperate with anti-LcrV to promote protection against lethal Y. pestis infection in mice. It is not known if these phagocyte-activating cytokines cooperate with anti-LcrV to increase the killing of the pathogen and decrease apoptosis in macrophages. We investigated how anti-LcrV and IFN-γ impact bacterial survival and apoptosis in cultured murine macrophages infected with Y. pestis KIM5. Y. pestis KIM5 opsonized with polyclonal or monoclonal anti-LcrV was used to infect macrophages treated with or without IFN-γ. The phagocytosis and survival of KIM5 and the apoptosis of macrophages were measured at different time points postinfection. The results show that anti-LcrV reduced apoptosis at an early time point (5 h) but not at a later time point (24 h). Polyclonal anti-LcrV was unable to inhibit apoptosis at either time point in IFN-γ-activated macrophages. Additionally, anti-LcrV was ineffective at promoting the killing of KIM5 in naïve or activated macrophages. We conclude that Y. pestis can bypass protective antibodies to LcrV and activation with IFN-γ to survive and induce apoptosis in murine macrophages.

Yersinia pestis continues to pose a threat as a potential biological weapon and is recognized by public health experts as a re-emerging infectious disease. Therefore there is great interest in developing a safe and effective vaccine. Vaccines against plague containing both the Fraction 1 (F1) and V antigens of Y. pestis have shown promise in protecting animal models against pneumonic plague, the deadliest form of the disease. Here we report on a plague vaccine consisting of the F1 and LcrV antigens fused to a single carrier molecule, the thermostable enzyme lichenase from Clostridium thermocellum, and expressed in and purified from Nicotiana benthamiana plants. When administered to Cynomolgus Macaques this purified plant-produced vaccine induced high titers of serum IgG, mainly of the IgG1 isotype, against both F1 and LcrV. These immunized animals were subsequently challenged and the LcrV-F1 plant-produced vaccine conferred complete protection against aerosolized Y. pestis.

The live plague vaccine strain Y. pestis EV line NIIEG widely used for human and animal immunisation proves a high level of specific immunity against both bubonic and pneumonic plague. We constructed an EV NIIEG mutant in the acyltransferase gene lpxM that resulted in the production of a less toxic penta-acylated lipid A of lipopolysaccharide (LPS) of Y. pestis , tested the synthesis and immunoreactivity of the major known antigens involved in virulence and eliciting immunity against plague, and also evaluated the protective properties of the lpxM derivative. The expression of antigens was determined by dot-ELISA, immunoblotting, the indirect hemagglutination test, electron microscopy (capsule), and fibrinolytic and plasma-coagulase activities (plasminogen activator Pla). Stumulation of TNF-alpha production with different types of LPS was revealed by using the J774 macrophage-like cell line. The antibody immune response, followed by immunisation, was estimated by ELISA. Protective efficacy was evaluated in mice and guinea pigs by subcutaneous challenge with the wild-type Y. pestis 231. In comparison with the parental strain, the lpxM mutant was found to produce a reduced amount of capsular antigen F1, multifunctional virulence factor LcrV and murine toxin Ymt as well as had an altered activity of Pla. Moreover, this mutant possessed a modified cell surface as jusjed by the immunoreactivity with monoclonal antibodies to different carbohydrate epitopes and by the resistant phenotype to the plague diagnostic bacteriophage L-413C. The penta-acylated LPS of Y. pestis revealed a reduced capacity of stimulating TNF-alpha in vitro . Immunisation with the lpxM mutant provided an enhanced protection over the original Y. pestis EV NIIEG in both mice and guinea pigs. Thus, the pleiotropic effect of the lpxM mutation led to the construction of the live plague candidate vaccine strain with improved characteristics toward safety, reactogenicity and protective properties.

Yersinia pestis causes pneumonic plague, an exceptionally virulent disease for which we lack a safe and effective vaccine. Antibodies specific for the Y. pestis F1 and LcrV proteins can protect mice against pulmonary Y. pestis infection. We demonstrate that neutralizing tumor necrosis factor-alpha (TNFalpha) and gamma-interferon (IFNgamma) abrogates this protection at sub-optimal levels of F1- or LcrV-specific antibody, but not at optimal levels. Moreover, we demonstrate that endogenous TNFalpha and IFNgamma confer measurable protection in the complete absence of protective antibodies. These findings indicate that antibodies and cytokines independently protect against pneumonic plague and suggest that surrogate assays for plague vaccine efficacy should consider both the level of vaccine-induced antibody and the capacity of vaccine recipients to produce TNFalpha and IFNgamma upon exposure to Y. pestis.

We have developed an experimental recombinant vesicular stomatitis virus (VSV) vectored plague vaccine expressing a secreted form of Yersinia pestis low calcium response protein V (LcrV) from the first position of the VSV genome. This vector, given intramuscularly in a single dose, induced high-level antibody titers to LcrV and gave 90–100% protection against pneumonic plague challenge in mice. This single-dose protection was significantly better than that generated by VSV expressing the non-secreted LcrV protein. Increased protection correlated with increased anti-LcrV antibody and a bias toward IgG2a and away from IgG1 isotypes. We also found that the depletion of CD4+ cells, but not CD8+ cells, at the time of challenge resulted in reduced vaccine protection, indicating a role for cellular immunity in protection.

Bubonic plague is the most devastating acute infectious disease known to man. The causative agent, Yersinia pestis, is now more firmly entrenched in sylvatic reservoirs throughout the world than at any time in the past. Consequently, the organism increasingly causes casual human disease and is readily available for use as a bioweapon. Recent attempts to understand the severe nature of plague have focused upon its very recent divergence from Yersinia pseudotuberculosis, an etiological instrument of chronic enteropathogenic infection. This review emphasizes that the invasive nature of plague and its dissemination by fleabite is mediated by plasmids not shared by enteropathogenic yersiniae. The basis for high lethality is considered within the context of chromosomal degeneration causing loss of normal metabolic functions and modification of virulence factors, permitting a terminal anti-inflammatory phase associated with pronounced septicemia.

Production of vaccine antigens in plants has received considerable attention over the last decade. However, despite many antigens being expressed in plant systems, and promising efficacy data with rodent models, few vaccine candidates have advanced into studies in non-human primates or human clinical trials. Here, we report on the transient expression of the F1 and LcrV antigens of Yersinia pestis in Nicotiana benthamiana. The antigens were expressed as fusions to the thermostable enzyme of Clostridium thermocellum. When administered to Cynomolgus Macaques the purified plant-produced antigens induced serum IgG and IgA responses specific to F1 and LcrV, and conferred complete protection against lethal challenge with Y. pestis. This study clearly demonstrates the efficacy of a plant-produced plague vaccine candidate in a primate model.

Vaccination with live attenuated Yersinia pestis confers protection against pneumonic plague but is not considered safe for general use. Subunit plague vaccines containing the Y. pestis F1 and LcrV proteins prime robust antibody responses but may not provide sufficient protection. To aid the development of a safe and effective plague vaccine, we are investigating roles for T cells during defense against Y. pestis infection. Here we demonstrate that vaccination of mice with live Y. pestis primes specific CD4 and CD8 T cells that, upon purification and direct transfer to naïve mice, synergistically protect against lethal intranasal Y. pestis challenge. While not preventing extrapulmonary dissemination, the coadministered T cells promote bacterial clearance and reduce bacteremia. These observations strongly suggest that development of pneumonic plague vaccines should strive to prime both CD4 and CD8 T cells. Finally, we demonstrate that vaccination with live Y. pestis primes CD4 and CD8 T cells that respond to Y. pestis strains lacking the capacity to express F1, LcrV, and all pCD1/pPCP-encoded proteins, suggesting that protective T cells likely recognize antigens distinct from those previously defined as targets for humoral immunity.