Protection against anthrax and plague by a combined vaccine in mice and rabbits

The protective antigen (PA) of Bacillus anthracis and the Fraction 1 Capsular Antigen (F1 antigen), V antigen of Yersinia pestis have been demonstrated to be potential immunogens and candidate vaccine sub-units against anthrax and plague respectively. In this study, the authors have investigated the antibody responses and the protective efficacy when the antigens were administered separately or in combination intramuscularly formulation adsorbed to an aluminum hydroxide adjuvant. Results show that immunized rF1 + rV and rPA antigen together was as effective as separately for induction of serological antibody response, and these titers were maintained for over 1 year in mice. An isotype analysis of the serum indicates that the co-administration of these antigens did not influence the antigen-specific IgG1/IgG2a ratio which was consistent with a Th2 bias. Furthermore, the combined vaccine comprising the protein antigens rF1 + rV + rPA has been demonstrated to protect mice from subcutaneous challenge with 10⁷ colony-forming units (CFU) virulent Y. pestis strain, and to fully protect rabbit against subcutaneous challenge with 1.2 × 10⁵ colony-forming units (CFU) virulent B. anthracis spores. These data show that the protective efficacy was unaffected when the antigens were administered in combination.     

Intranasal delivery of a protein subunit vaccine using a Tobacco Mosaic Virus platform protects against pneumonic plague

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.     

Comparison of mouse,guinea pig and rabbit models for evaluation of plague subunit vaccine F1 + rV270

In this study, a new subunit vaccine that comprised native F1 and recombinant rV270 was evaluated for protective efficacy using mouse, guinea pig and rabbit models in comparison with the live attenuated vaccine EV76. Complete protection against challenging with 10⁶ colony-forming units (CFU) of virulent Yersinia pestis strain 141 was observed for mice immunized with the subunit vaccines and EV76 vaccine. In contrast, the subunit vaccine recipes VII (F1-20 μg + rV270-10 μg) and IX (F1-40 μg + rV270-20 μg) and EV76 vaccine provided 86%, 79% and 93% protection against the same level of challenge in guinea pigs and 100%, 83% and 100% protection in rabbits, respectively. The immunized mice with the vaccines had significantly higher IgG titres than the guinea pigs and rabbits, and the immunized guinea pigs developed significantly higher IgG titres than the rabbits, but the anti-F1 response in guinea pigs was more variable than in the mice and rabbits, indicating that guinea pig is not an ideal model for evaluating protective efficacy of plague subunit vaccine, instead the rabbits could be used as an alternative model. All the immunized animals with EV76 developed a negligible IgG titre to rV270 antigen. Furthermore, analysis of IgG subclasses in the immunized animals showed a strong response for IgG1, whereas those receiving EV76 immunization demonstrated predominant production of IgG1 and IgG2a isotypes. The subunit vaccine and EV76 vaccine are able to provide protection for animals against Y. pestis challenge, but the subunit vaccines have obvious advantages over EV76 in terms of safety of use.     

Protection in mice passively immunized with serum from cynomolgus macaques and humans vaccinated with recombinant plague vaccine (rF1V)

Passive transfer models were developed to evaluate the ability of antibodies generated in cynomolgus macaques and humans vaccinated with a recombinant plague vaccine (rF1V) to protect naïve Swiss Webster mice against pneumonic plague. Development of the passive transfer model is intended to support clinical and nonclinical development of the rF1V vaccine. To evaluate protection, unfractionated serum collected from rF1V vaccinated cynomolgus macaques and human volunteers with known antibody titers to rF1, rV and rF1V was transferred into naïve Swiss Webster mice via the intraperitoneal route. Results of these studies demonstrated that passive immunization protected mice from challenge or extended mean survival time and that the passive transfer assay can be used to evaluate the functional role of antibodies induced by rF1V vaccination in protection against aerosol exposure.     

Robust Th1 cellular and humoral responses generated by the Yersinia pestis rF1-V subunit vaccine formulated to contain an agonist of the CD137 pathway do not translate into increased protection against pneumonic plague

Yersinia pestis is the causative agent of plague and is a re-emerging pathogen that also has the potential as a biological weapon, necessitating the development of a preventive vaccine. Despite intense efforts for the last several decades, there is currently not a vaccine approved by the FDA. The rF1-V vaccine adjuvanted with Alhydrogel is a lead candidate subunit vaccine for plague and generates a strong Th2-mediate humoral response with a modest Th1 cellular response. As immune protection against Y. pestis requires both humoral and Th1 cellular responses, modifying the rF1-V subunit vaccine formulation to include a robust inducer of Th1 responses may improve efficacy. Thus, we reformulated the subunit vaccine to include SA-4-1BBL, an agonist of the CD137 costimulatory pathway and a potent inducer of Th1 response, and assessed its protective efficacy against pneumonic plague. We herein show for the first time a sex bias in the prophylactic efficacy of the Alhydrogel adjuvanted rF1-V vaccine, with female mice showing better protection against pneumonic plague than male. The sex bias for protection was irrespective of the generation of comparable levels of rF1-V-specific antibody titers and Th1 cellular responses in both sexes. The subunit vaccine reformulated with SA-4-1BBL generated robust Th1 cellular and humoral responses. A prime-boost vaccination scheme involving prime with rF1-V + Alhydrogel and boost with the rF1-V + SA-4-1BBL provided protection in male mice against pneumonic plague. In marked contrast, prime and boost with rF1-V reformulated with both adjuvants resulted in the loss of protection against pneumonic plague, despite generating high levels of humoral and Th1 cellular responses. While unexpected, these findings demonstrate the complexity of immune mechanisms required for protection. Elucidating mechanisms responsible for these differences in protection will help to guide the development of better prophylactic subunit vaccines effective against pneumonic plague.     

Prevention of pneumonic plague in mice, rats, guinea pigs and non-human primates with clinical grade rV10, rV10-2 or F1-V vaccines

Yersinia pestis causes plague, a disease with high mortality in humans that can be transmitted by fleabite or aerosol. A US Food and Drug Administration (FDA)-licensed plague vaccine is currently not available. Vaccine developers have focused on two subunits of Y. pestis: LcrV, a protein at the tip of type III secretion needles, and F1, the fraction 1 pilus antigen. F1-V, a hybrid generated via translational fusion of both antigens, is being developed for licensure as a plague vaccine. The rV10 vaccine is a non-toxigenic variant of LcrV lacking residues 271-300. Here we developed Current Good Manufacturing Practice (cGMP) protocols for rV10. Comparison of clinical grade rV10 with F1-V did not reveal significant differences in plague protection in mice, guinea pigs or cynomolgus macaques. We also developed cGMP protocols for rV10-2, a variant of rV10 with an altered affinity tag. Immunization with rV10-2 adsorbed to aluminum hydroxide elicited antibodies against LcrV and conferred pneumonic plague protection in mice, rats, guinea pigs, cynomolgus macaques and African Green monkeys. The data support further development of rV10-2 for FDA Investigational New Drug (IND) authorization review and clinical testing.     

A recombinant bivalent fusion protein rVE confers active and passive protection against Yersinia enterocolitica infection in mice

In the present study, a bivalent chimeric protein rVE comprising immunologically active domains of Yersinia pestis LcrV and YopE was assessed for its prophylactic abilities against Yersinia enterocolitica O:8 infection in murine model. Mice immunized with rVE elicited significantly higher antibody titers with substantial contribution from the rV component (3:1 ratio). Robust and significant resistance to Y. enterocolitica infection with 100% survival (P < 0.001) was seen in rVE vaccinated mice when intra peritoneal (I.P.) challenged with 10⁸ CFU of Y. enterocolitica O:8 against the 75%, 60% and 75% survival seen in mice immunized with rV, rE, rV + rE, respectively. Macrophage monolayer supplemented with anti-rVE polysera illustrated efficient protection (89.41% survival) against challenge of Y. enterocolitica O:8. In contrast to sera from sham-immunized mice, immunization with anti-rVE polysera provided complete protection to BALB/c mice against I.P. challenge with 10⁸ CFU of Y. enterocolitica O:8 and developed no conspicuous signs of infection in necropsy. The histopathological analysis of microtome sections confirmed significantly reduced lesion size or no lesion in liver and intestine upon infection in anti-rVE immunized mice. The findings from this study demonstrated the fusion protein rVE as a potential candidate subunit vaccine and showed the functional role of antibodies in protection against Y. enterocolitica infections.     

T cells play an essential role in anti-F1 mediated rapid protection against bubonic plague

Plague, which is initiated by Yersinia pestis infection, is a fatal disease that progresses rapidly and leads to high mortality rates if not treated. Antibiotics are an effective plague therapy, but antibiotic-resistant Y. pestis strains have been reported and therefore alternative countermeasures are needed. In the present study, we assessed the potential of an F1 plus LcrV-based vaccine to provide protection shortly pre- or post-exposure to a lethal Y. pestis infection.Mice vaccinated up to one day before or even several hours after subcutaneous challenge were effectively protected. Mice immunized one or three days pre-challenge were protected even though their anti-F1 and anti-LcrV titers were below detection levels at the day of challenge. Moreover, using B-cell deficient μMT mice, we found that rapidly induced protective immunity requires the integrity of the humoral immune system. Analysis of the individual contributions of vaccine components to protection revealed that rF1 is responsible for the observed rapid antibody-mediated immunity. Applying anti-F1 passive therapy in the mouse model of bubonic plague demonstrated that anti-F1 F(ab′)₂ can delay mortality, but it cannot provide long-lasting protection, as do intact anti-F1 molecules. Fc-dependent immune components, such as the complement system and (to a lesser extent) neutrophils, were found to contribute to mouse survival. Interestingly, T cells but not B cells were found to be essential for the recovery of infected animals following passive anti-F1 mediated therapy. These data extend our understanding of the immune mechanisms required for the development of a rapid and effective post-exposure therapy against plague.     

Small protective fragments of the Yersinia pestis V antigen

Yersinia pestis is the causative agent of plague. Naturally occurring cases of the disease and the potential use of Y. pestis as a bioweapon fuel the need for efficacious vaccines. The most recent plague vaccine is a killed whole cell preparation that is expensive to manufacture and its side effects are common. The protective antigens F1 and V have been identified and are currently being developed as a combined subunit vaccine. Protective epitopes of the V antigen have previously been shown to reside in the central part of the protein. In order to identify the minimum protective fragment of the V antigen that can provide protection against plague, the structures of several small fragments of the antigen were modelled in silico and recombinant proteins were produced. These fragments were probed for the retention of a protective epitope using a protective monoclonal antibody and protection against Y. pestis in mice was determined. The smallest protective fragment of V antigen identified comprised amino acids 135–262. Finally the ability of this fragment to confer protection when given in the context of a DNA vaccine was confirmed.     

Further development of raccoon poxvirus-vectored vaccines against plague (Yersinia pestis)

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.     

Protection against pneumonic plague following oral immunization with a non-replicating vaccine

Yersinia pestis is a dangerous bacterial pathogen that when inhaled can rapidly induce fatal pneumonic plague. Thus, there is a need for stable, safe, and easily administered mucosal vaccines capable of eliciting effective protection against pulmonary Y. pestis infections. Cationic liposome–nucleic acid complexes (CLDC) have been shown previously to be effective vaccine adjuvants for parenteral immunization, but have not been previously evaluated for use in oral immunization. Therefore, we investigated the ability of an orally administered CLDC adjuvanted vaccine to elicit protective immunity against lethal pneumonic plague. C57Bl/6 mice were vaccinated orally or subcutaneously using 10 μg Y. pestis F1 antigen combined with CLDC and immune responses and protection from challenge was assessed. We found that oral immunization elicited high titers of anti-F1 antibodies, equivalent to those generated by parenteral immunization. Importantly, orally immunized mice were protected from lethal pulmonary challenge with virulent Y. pestis for up to 18 weeks following vaccination. Vaccine-induced protection following oral immunization was found to be dependent primarily on CD4+ T cells, with a partial contribution from CD8+ T cells. Thus, CLDC adjuvanted vaccines represent a new type of orally administered, non-replicating vaccine capable of generating effective protection against pulmonary infection with virulent Y. pestis.     

Efficacy and safety of a modified vaccinia Ankara (MVA) vectored plague vaccine in mice

The efficacy and safety of plague vaccines based on the modified vaccinia Ankara (MVA) viral vector was evaluated. MVA recombinants were constructed expressing Yersinia pestis antigens under the translational control of the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and/or fused to the tissue plasminogen activator (tPA) secretory signal. A MVA/Y. pestis recombinant that expressed a truncated version of the low-calcium response V antigen (MVA/IRES/tPA/V₃₀₇), conferred significant protection (87.5–100%) against intranasal or intraperitoneal challenge with CO92 (encapsulated) or Java 9 (non-encapsulated) strains of Y. pestis, respectively. In contrast, a MVA/Y. pestis recombinant that expressed the full-length V antigen provided only 37.5% protection against challenge with CO92 or Java 9 strains, respectively. Interestingly, a MVA/Y. pestis recombinant that expressed the capsular protein (F1) did not elicit significant antibody titers but still conferred 50% and 25% protection against CO92 or Java 9 challenge, respectively. The MVA/Y. pestis recombinant viruses did not demonstrate any mortality or morbidity in SCID mice. Based on their safety and efficacy in mice, these MVA/Y. pestis recombinants are candidates for further development as biodefense and public health vaccines.     

Amino acid residues 196–225 of LcrV represent a plague protective epitope

LcrV, a protein that resides at the tip of the type III secretion needles of Yersinia pestis, is the single most important plague protective antigen. Earlier work reported monoclonal antibody MAb 7.3, which binds a conformational epitope of LcrV and protects experimental animals against lethal plague challenge. By screening monoclonal antibodies directed against LcrV for their ability to protect immunized mice against bubonic plague challenge, we examined here the possibility of additional protective epitopes. MAb BA5 protected animals against plague, neutralized the Y. pestis type III secretion pathway and promoted opsonophagocytic clearance of bacteria in blood. LcrV residues 196–225 were necessary and sufficient for MAb BA5 binding. Compared to full-length LcrV, a variant lacking its residues 196–225 retained the ability of eliciting plague protection. These results identify LcrV residues 196–225 as a linear epitope that is recognized by the murine immune system to confer plague protection.     

Quantitative anti-F1 and anti-V IgG ELISAs as serological correlates of protection against plague in female Swiss Webster mice

A recombinant fusion protein composed of Yersinia pestis fraction 1 capsule (F1) and virulence-associated V antigen (V) (F1–V) has been developed as the next-generation vaccine against plague. In this study, female Swiss Webster mice received a single intramuscular vaccination with one of eight doses of the F1–V vaccine and exposed 4 weeks later to either Y. pestis CO92 or C12 organisms by the subcutaneous or aerosol routes of infection. Quantitative anti-F1 and anti-V immunoglobulin G (IgG) ELISAs were used to examine the relationship between survival outcome and antibody titers to F1 and V. Results suggested that each 1 log₁₀ increase in week 4 quantitative anti-F1 and anti-V IgG ELISA titers were associated with a 1.7-fold (p = 0.0051) and 2.5-fold (p = 0.0054) increase in odds of survival, respectively, against either bubonic or pneumonic plague and may serve as serological correlates of protection.     

A live attenuated strain of Yersinia pestis KIM as a vaccine against plague

Yersinia pestis, the causative agent of plague, is a potential weapon of bioterrorism. Y. pestis evades the innate immune system by synthesizing tetra-acylated lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity at 37 °C, whereas hexa-acylated lipid A, a potent TLR4 agonist, is made at lower temperatures. Synthesis of Escherichia coli LpxL, which transfers the secondary laurate chain to the 2′-position of lipid A, in Y. pestis results in production of hexa-acylated lipid A at 37 °C, leading to significant attenuation of virulence. Previously, we described a Y. pestis vaccine strain in which crp expression is under the control of the arabinose-regulated araC P_BAD promoter, resulting in a 4-5 log reduction in virulence. To reduce the virulence of the crp promoter mutant further, we introduced E. coli lpxL into the Y. pestis chromosome. The χ10030(pCD1Ap) (ΔlpxP32::P_lpxLlpxL ΔP_crp21::TT araC P_BADcrp) construct likewise produced hexa-acylated lipid A at 37 °C and was significantly more attenuated than strains harboring each individual mutation. The LD₅₀ of the mutant in mice, when administered subcutaneously or intranasally was >10⁷-times and >10⁴-times greater than wild type, respectively. Mice immunized subcutaneously with a single dose of the mutant were completely protected against a subcutaneous challenge of 3.6 × 10⁷ wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2 × 10⁴ live cells. Intranasal immunization also provided significant protection against challenges by both routes. This mutant is an immunogenic, highly attenuated live Y. pestis construct that merits further development as a vaccine candidate.     

TNFα and IFNγ contribute to F1/LcrV-targeted immune defense in mouse models of fully virulent pneumonic plague

Immunization with the Yersinia pestis F1 and LcrV proteins improves survival in mouse and non-human primate models of pneumonic plague. F1- and LcrV-specific antibodies contribute to protection, however, the mechanisms of antibody-mediated defense are incompletely understood and serum antibody titers do not suffice as quantitative correlates of protection. Previously we demonstrated roles for tumor necrosis factor-alpha (TNFα) and gamma-interferon (IFNγ) during defense against conditionally attenuated pigmentation (pgm) locus-negative Y. pestis. Here, using intranasal challenge with fully virulent pgm-positive Y. pestis strain CO92, we demonstrate that neutralizing TNFα and IFNγ interferes with the capacity of therapeutically administered F1- or LcrV-specific antibody to reduce bacterial burden and increase survival. Moreover, using Y. pestis strain CO92 in an aerosol challenge model, we demonstrate that neutralizing TNFα and IFNγ interferes with protection conferred by immunization with recombinant F1-LcrV fusion protein vaccine (p < 0.0005). These findings establish that TNFα and IFNγ contribute to protection mediated by pneumonic plague countermeasures targeting F1 and LcrV, and suggest that an individual's capacity to produce these cytokines in response to Y. pestis challenge will be an important co-determinant of antibody-mediated defense against pneumonic plague.     

Development of a vaccinia virus based reservoir-targeted vaccine against Yersinia pestis

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.     

Evaluation of Psn, HmuR and a modified LcrV protein delivered to mice by live attenuated Salmonella as a vaccine against bubonic and pneumonic Yersinia pestis challenge

We evaluated the ability of Yersinia pestis antigens HmuR, Psn and modified forms of LcrV delivered by live attenuated Salmonella strains to stimulate a protective immune response against subcutaneous or intranasal challenge with Y. pestis CO92. LcrV196 is a previously described truncated protein that includes aa 131-326 of LcrV and LcrV5214 has been modified to replace five key amino acids required for interaction with the TLR2 receptor. Psn is the outer membrane receptor for the siderophore, yersiniabactin, and the bacteriocin, pesticin. Mice immunized with Salmonella synthesizing Psn, LcrV196 or LcrV5214 developed serum IgG responses to the respective Yersinia antigen and were protected against pneumonic challenge with Y. pestis. Immunization with Salmonella synthesizing Psn or LcrV196 was sufficient to afford nearly full protection against bubonic challenge, while immunization with the strain synthesizing LcrV5214 was not protective. Immunization with Salmonella synthesizing HmuR, an outer membrane protein involved in heme acquisition in Y. pestis, was poorly immunogenic and did not elicit a protective response against either challenge route. These findings indicate that both Psn and LcrV196 delivered by Salmonella provide protection against both bubonic and pneumonic plague.     

Multiple antigens of Yersinia pestis delivered by live recombinant attenuated Salmonella vaccine strains elicit protective immunity against plague

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.     

Involvement of CD8+ T cell-mediated immune responses in LcrV DNA vaccine induced protection against lethal Yersinia pestis challenge

Yersinia pestis (Y. pestis) is the causative pathogen of plague, a highly fatal disease for which an effective vaccine, especially against mucosal transmission, is still not available. Like many bacterial infections, antigen-specific antibody responses have been traditionally considered critical, if not solely responsible, for vaccine-induced protection against Y. pestis. Studies in recent years have suggested the importance of T cell immune responses against Y. pestis infection but information is still limited about the details of Y. pestis antigen-specific T cell immune responses. In current report, studies are conducted to identify the presence of CD8+ T cell epitopes in LcrV protein, the leading antigen of plague vaccine development. Furthermore, depletion of CD8+ T cells in LcrV DNA vaccinated Balb/C mice led to reduced protection against lethal intranasal challenge of Y. pestis. These findings establish that an LcrV DNA vaccine is able to elicit CD8+ T cell immune responses against specific epitopes of this key plague antigen and that a CD8+ T cell immune response is involved in LcrV DNA vaccine-elicited protection. Future studies in plague vaccine development will need to examine if the presence of detectable T cell immune responses, in particular CD8+ T-cell immune responses, will enhance the protection against Y. pestis in higher animal species or humans.