Evaluation of Protective Potential of Yersinia pestis Outer Membrane Protein Antigens as Possible Candidates for a New-Generation Recombinant Plague Vaccine

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.     

Direct Neutralization of Type III Effector Translocation by the Variable Region of a Monoclonal Antibody to Yersinia pestis LcrV

Plague is an acute infection caused by the Gram-negative bacterium Yersinia pestis. Antibodies that are protective against plague target LcrV, an essential virulence protein and component of a type III secretion system of Y. pestis. Secreted LcrV localizes to the tips of type III needles on the bacterial surface, and its function is necessary for the translocation of Yersinia outer proteins (Yops) into the cytosol of host cells infected by Y. pestis. Translocated Yops counteract macrophage functions, for example, by inhibiting phagocytosis (YopE) or inducing cytotoxicity (YopJ). Although LcrV is the best-characterized protective antigen of Y. pestis, the mechanism of protection by anti-LcrV antibodies is not fully understood. Antibodies bind to LcrV at needle tips, neutralize Yop translocation, and promote opsonophagocytosis of Y. pestis by macrophages in vitro. However, it is not clear if anti-LcrV antibodies neutralize Yop translocation directly or if they do so indirectly, by promoting opsonophagocytosis. To determine if the protective IgG1 monoclonal antibody (MAb) 7.3 is directly neutralizing, an IgG2a subclass variant, a deglycosylated variant, F(ab′)₂, and Fab were tested for the ability to inhibit the translocation of Yops into Y. pestis-infected macrophages in vitro. Macrophage cytotoxicity and cellular fractionation assays show that the Fc of MAb 7.3 is not required for the neutralization of YopJ or YopE translocation. In addition, the use of Fc receptor-deficient macrophages, and the use of cytochalasin D to inhibit actin polymerization, confirmed that opsonophagocytosis is not required for MAb 7.3 to neutralize translocation. These data indicate that the binding of the variable region of MAb 7.3 to LcrV is sufficient to directly neutralize Yop translocation.     

LcrV Capture Enzyme-Linked Immunosorbent Assay for Detection of Yersinia pestis from Human Samples

In the United States, there is currently a major gap in the diagnostic capabilities with regard to plague. To address this, we developed an antigen capture assay using an essential virulence factor secreted by Yersinia spp., LcrV, as the target antigen. We generated anti-LcrV monoclonal antibodies (MAbs) and screened them for the ability to bind bacterially secreted native Yersinia pestis LcrV. Anti-LcrV MAb 19.31 was used as a capture antibody, and biotinylated MAb 40.1 was used for detection. The detection limit of this highly sensitive Yersinia LcrV capture enzyme-linked immunosorbent assay is 0.1 ng/ml. The assay detected LcrV from human sputum and blood samples treated with concentrations as low as 0.5 ng/ml of bacterially secreted native Y. pestis LcrV. This assay could be used as a tool to help confirm the diagnosis of plague in patients presenting with pneumonia.     

Acquisition of Maternal Antibodies both from the Placenta and by Lactation Protects Mouse Offspring from Yersinia pestis Challenge

Artificially passive immunization has been demonstrated to be effective against Yersinia pestis infection in animals. However, maternal antibodies'' protective efficacy against plague has not yet been demonstrated. Here, we evaluated the kinetics, protective efficacy, and transmission modes of maternal antibodies, using mice immunized with plague subunit vaccine SV1 (20 μg of F1 and 10 μg of rV270). The results showed that the rV270- and F1-specific antibodies could be detected in the sera of newborn mice (NM) until 10 and 14 weeks of age, respectively. There was no antibody titer difference between the parturient mice immunized with SV1 (PM-S) and the caesarean-section newborns (CSN) from the PM-S or between the lactating mice immunized by SV1 (LM-S) and the cross-fostered mice (CFM) during 3 weeks of lactation. The NM had a 72% protection against 4,800 CFU Y. pestis strain 141 challenge at 6 weeks of age, whereas at 14 weeks of age, NM all succumbed to 5,700 CFU of Y. pestis challenge. After 7 weeks of age, CFM had an 84% protection against 5,000 CFU of Y. pestis challenge. These results indicated that maternal antibodies induced by the plague subunit vaccine in mother mice can be transferred to NM by both placenta and lactation. Passive antibodies from the immunized mothers could persist for 3 months and provide early protection for NM. The degree of early protection is dependent on levels of the passively acquired antibody. The results indicate that passive immunization should be an effective countermeasure against plague during its epidemics.     

D27-pLpxL,an Avirulent Strain of Yersinia pestis,Primes T Cells That Protect against Pneumonic Plague

Vaccinating with live, conditionally attenuated, pigmentation (Pgm)-deficient Yersinia pestis primes T cells that protect mice against pneumonic plague. However, Pgm-deficient strains are not considered safe for human use because they retain substantial virulence in animal models. Y. pestis strains engineered to express Escherichia coli LpxL are avirulent owing to constitutive production of lipopolysaccharide with increased Toll-like receptor 4-activating ability. We generated an LpxL-expressing Pgm-deficient strain (D27-pLpxL) and demonstrate here that this avirulent strain retains the capacity to prime protective T cells. Compared with unvaccinated controls, mice immunized intranasally with live D27-pLpxL exhibit a decreased bacterial burden and increased survival when challenged intranasally with virulent Y. pestis. T cells provide a substantial degree of this protection, as vaccine efficacy is maintained in B-cell-deficient μMT mice unless those animals are depleted of CD4 and CD8 T cells at the time of challenge. Upon challenge with Y. pestis, pulmonary T-cell numbers decline in naive mice, whereas immunized mice show increased numbers of CD44^high CD43^high effector T cells and T cells primed to produce tumor necrosis factor alpha and gamma interferon; neutralizing these cytokines at the time of challenge abrogates protection. Immunization does not prevent dissemination of Y. pestis from the lung but limits bacterial growth and pathology in visceral tissue, apparently by facilitating formation of granuloma-like structures. This study describes a new model for studying T-cell-mediated protection against pneumonic plague and demonstrates the capacity for live, highly attenuated, Y. pestis vaccine strains to prime protective memory T-cell responses safely.Plague, also known as the Black Death, has killed hundreds of millions of people over the course of three major pandemics (33). Yersinia pestis, the etiologic agent of plague, is a gram-negative facultative intracellular bacterium naturally transmitted to mammals by the bite of an infected flea (31). There are three clinical forms of Y. pestis infection: bubonic, septicemic, and pneumonic plague (5). The bubonic form is characterized by a swelling of lymph nodes draining the fleabite. Left untreated, bubonic plague typically progresses to bacteremia and septicemia. The pneumonic form of plague, which can spread from person to person without an intermediary insect vector, occurs when Y. pestis colonizes the lung, either as a secondary consequence of bacteremia or via direct inhalation of infectious bacilli. Unless treated with antibiotics soon after symptom onset, all forms of plague can be fatal. Pneumonic plague is particularly fulminant and often is lethal even with antibiotic treatment (5, 20, 23).Armed with knowledge of the cause of plague and its routes of transmissibility, public health efforts have substantially reduced the likelihood of a modern-day pandemic. However, sporadic plague outbreaks continue to afflict human populations (47), and Y. pestis is now entrenched in rodent populations on nearly every continent (7). Moreover, Y. pestis displays significant clonal diversity, and some strains already resist treatment with multiple antibiotics (13, 19). In addition to these natural threats, there is concern that Y. pestis may be exploited as a biological weapon (19). Accordingly, substantial resources are being devoted to the development of plague countermeasures.The search for a safe and effective plague vaccine began shortly after Yersin identified the causative agent (48). Immunization with killed “whole-cell” Y. pestis bacilli protects humans against bubonic plague, but vaccines of this type are reactogenic, require frequent boosting, and are not thought to protect humans from pneumonic plague (5, 24, 43). Subunit vaccines comprised of the Y. pestis F1 and/or LcrV protein appear to be safe and well tolerated by humans (46). These vaccines provide substantial protection in animal models of pneumonic plague (38, 43). However, the F1/LcrV-based vaccines have failed, thus far, to fully protect nonhuman primates, and we lack adequate knowledge about immune correlates of protection to confidently predict whether or not they will protect humans (3, 38).Administering monoclonal antibodies (MAbs) specific for F1 or LcrV can protect mice against pneumonic challenge with fully virulent Y. pestis (2, 15, 16). Given this documented efficacy of antibody-mediated defense, most F1/LcrV vaccine efforts have aimed to induce high-titer antibodies. However, bioweaponeers potentially could circumvent vaccines based solely on F1 and LcrV, since F1-deficient strains retain virulence and LcrV variants exist (38, 43). Moreover, a number of studies suggest that antibodies alone may not provide optimal protection against pneumonic plague (39). First, levels of antibodies in F1/LcrV-immunized mice and nonhuman primates often fail to correlate with protective efficacy (3). Second, F1/LcrV-immunized STAT4-deficient mice are poorly protected against plague challenge, despite possessing high antibody titers (8). STAT4-deficient mice lack the capacity to mount robust type 1 immune responses, and our recent studies indicate that the protection mediated by F1- and LcrV-specific antibody benefits from gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), cytokine products of type 1 immunity (21). Chattopadhyay et al. recently demonstrated that protection conferred by immunizing mice with an LcrV-expressing viral vector benefits from the presence of CD4 T cells at the time of challenge (6). Together, these observations raise the possibility that high-titer antibody may not suffice to protect humans from pneumonic plague and suggest that vaccines harnessing both humoral and cellular defense mechanisms should provide superior defense (38, 39).Live vaccines typically prime both antibody- and cell-mediated immune responses. Pigmentation (Pgm)-deficient strains of Y. pestis arise spontaneously during in vitro passage, usually owing to deletion of a 102-kb region of the chromosome that encodes proteins related to iron utilization (10). Immunization with Pgm-deficient strains, which are highly attenuated when administered subcutaneously (44), protects rodents against pneumonic plague (36). A Pgm-deficient vaccine strain, denoted EV76, was developed for human use nearly a century ago (14). It was administered to tens of millions of humans (14) and is still in use today in Russia (9, 50). However, live plague vaccines have never been licensed in the United States or the United Kingdom, in part because Pgm-deficient strains are only conditionally attenuated in mice, which tolerate high doses administered subcutaneously but succumb to disease at relatively low doses administered intravenously or intranasally (28, 29, 32, 44, 45). Moreover, primate species display various degrees of susceptibility (9, 25, 45).In a prior study we demonstrated that immunizing mice with live conditionally attenuated Pgm-deficient Y. pestis strain D27 primes T-cell-mediated protection against pneumonic challenge (28). Here we extend that observation to D27-pLpxL, a Pgm-deficient Y. pestis strain rendered avirulent by engineered constitutive expression of inflammatory forms of lipopolysaccharide. The protection conferred by this strain does not require antibody production and is associated with robust priming of cytokine-producing T cells that enable leukocytes to form granuloma-like structures and resist the cytopathic effects that typically characterize visceral Y. pestis infections. These findings suggest that it should be possible to generate safe, highly attenuated, live Y. pestis vaccines that prime protective memory T-cell responses.     

High-Throughput Identification of New Protective Antigens from a Yersinia pestis Live Vaccine by Enzyme-Linked Immunospot Assay

Yersinia pestis, the plague pathogen, is a facultative intracellular bacterium. Cellular immunity plays important roles in defense against infections. The identification of T-cell targets is critical for the development of effective vaccines against intracellular bacteria; however, the function of cellular immunity in protection from plague was not clearly understood. In this study, 261 genes from Y. pestis were selected on the basis of bioinformatics analysis and previous research results for expression in Escherichia coli BL21(DE3). After purification, 101 proteins were qualified for examination of their abilities to induce the production of gamma interferon in mice immunized with live vaccine EV76 by enzyme-linked immunospot assay. Thirty-four proteins were found to stimulate strong T-cell responses. The protective efficiencies for 24 of them were preliminarily evaluated using a mouse plague model. In addition to LcrV, nine proteins (YPO0606, YPO1914, YPO0612, YPO3119, YPO3047, YPO1377, YPCD1.05c, YPO0420, and YPO3720) may provide partial protection against challenge with a low dose (20 times the 50% lethal dose [20× LD₅₀]) of Y. pestis, but only YPO0606 could partially protect mice from infection with Y. pestis at a higher challenge dosage (200× LD₅₀). These proteins would be the potential components for Y. pestis vaccine development.Yersinia pestis is a category A pathogen and a potential agent of bioterrorism and biological warfare (6, 8). This gram-negative bacterium causes bubonic, septicemic, or pneumonic plague and has killed millions of people during the three major pandemics in history. Even today, at least 2,000 cases of plague are reported annually to the World Health Organization (WHO). Furthermore, the identification of natural antibiotic-resistant strains emphasizes that the development of an effective vaccine is one of the urgent needs in the prevention of Y. pestis infections (15, 16).The great efforts in many laboratories have made promising progress in the development of vaccines against both bubonic and pneumonic plague (7, 20, 41). Until now, most of these studies have focused largely upon antibody-based humoral immunity. However, results from vaccine trials with nonhuman primates suggest that humoral immunity may not suffice to protect humans against Y. pestis infection. Several studies of animal models have confirmed the roles of CD4⁺ T helper 1 (Th1) cells and the cytokines gamma interferon (IFN-γ) and tumor necrosis factor alpha in plague protection (24, 32, 34, 40). Cellular and humoral immune responses will synergize in combating plague infection. Ideally, plague vaccines should elicit both cellular and humoral immunity.The subunit vaccines based on F1 (the fraction 1 capsule-like antigen) and/or V (LcrV) antigen proved to be efficient in small animals under laboratory conditions, and both of them can induce antibody responses with high titers (38, 47). However, there is no a priori reason to assume that F1 and/or V constitutes the immunodominant target for the T-cell response, although previous findings indicate that Y. pestis must possess antigenic targets for cellular immunity (34). Thus, to effectively incorporate cellular immunity into plague subunit vaccines, it is now imperative to define the specific Y. pestis proteins that elicit cellular immune responses.The availability of complete bacterial genome sequences makes a push for reverse vaccinology to be put into practice (29, 31). The development of protein microarrays and immunoproteomics let us identify novel immunogens that induce humoral immune responses in a high-throughput manner (1, 12, 19, 21, 27, 28). The approach to identify T-cell antigens on a large scale is under way (10, 14).In this report, we describe the use of in silico computer-based analysis in combination with an in vitro IFN-γ assay to identify potential T-cell antigens from Y. pestis by a high-throughput approach. In total, 34 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with Y. pestis live vaccine EV76 were identified. Nine of them can provide partial protection against challenge with 20 times the 50% lethal dose (20× LD₅₀) of Y. pestis.     

The smpB-ssrA Mutant of Yersinia pestis Functions as a Live Attenuated Vaccine To Protect Mice against Pulmonary Plague Infection

The bacterial SmpB-SsrA system is a highly conserved translational quality control mechanism that helps maintain the translational machinery at full capacity. Here we present evidence to demonstrate that the smpB-ssrA genes are required for pathogenesis of Yersinia pestis, the causative agent of plague. We found that disruption of the smpB-ssrA genes leads to reduction in secretion of the type III secretion-related proteins YopB, YopD, and LcrV, which are essential for virulence. Consistent with these observations, the smpB-ssrA mutant of Y. pestis was severely attenuated in a mouse model of infection via both the intranasal and intravenous routes. Most significantly, intranasal vaccination of mice with the smpB-ssrA mutant strain of Y. pestis induced a strong antibody response. The vaccinated animals were well protected against subsequent lethal intranasal challenges with virulent Y. pestis. Taken together, our results indicate that the smpB-ssrA mutant of Y. pestis possesses the desired qualities for a live attenuated cell-based vaccine against pneumonic plague.Plague, a dangerous and often deadly disease, is caused by a Gram-negative bacterium, Yersinia pestis (38, 39). Depending on the route of entry, the disease can develop into a variety of forms, such as bubonic, pneumonic, or septicemic plague. Pneumonic plague is considered the most dangerous form of the disease since the organism can disseminate through aerosol droplets, resulting in high mortality. In fact, these features have led to the classification of Y. pestis as a category A agent of bioterrorism (24). Antibiotic therapy can be effective upon early diagnosis of plague. However, the appearance of multidrug-resistant Y. pestis strains in recent years presents a challenge for currently available antibiotic therapy (39). Therefore, there is a need for a safe and effective plague vaccine, which is currently not available.Animal infection studies have identified several antigens that could be used as recombinant subunit vaccines. These include the F1 antigen and the LcrV protein. Active or passive immunization of experimental animals with these antigens was shown to be protective against pneumonic plague (1-3, 18, 23). However, F1⁻ mutants of Y. pestis have been reported to retain full virulence in animal infection studies (15, 41, 52). Also, animals immunized with the LcrV protein can still be susceptible to Yersinia infections due to the variations in LcrV protein (44). Such strains could circumvent the effectiveness of subunit vaccines. Therefore, inclusion of additional elements, such as additional antigens or a library of antigens, could provide better protection against genetically engineered, fully virulent Y. pestis strains. One way to present many antigens at once is to utilize killed or live attenuated Y. pestis organisms. The use of heat-killed or formalin-fixed Y. pestis has a long history as a plague vaccine, and they were shown to be effective against bubonic plague (46). However, these vaccines have also caused significant adverse reactions, such as fever, malaise, headache, and lymphadenopathy. In addition, immunization with heat- or formalin-killed bacteria has generally failed to protect experimental animals against pneumonic plague (46). On the other hand, live attenuated plague vaccines, such as one based on the Y. pestis EV76 strain, appeared to be protective against pneumonic plague (46, 49, 53). Such genetically undefined strains can be unstable and retain significant virulence. Therefore, there is still a need to identify novel attenuated Y. pestis strains that can be used in production of safe and effective vaccines against all forms of plague.SsrA is a unique RNA molecule that performs an important quality control function in collaboration with its protein partner, SmpB (17). SsrA RNA functions as both tRNA and mRNA through its unique sequence and structural properties. The SmpB-SsrA function is required to deal with ribosomes stalled on defective mRNAs (27, 28). The smpB and ssrA genes are present in all bacteria examined to date (21, 28, 51). The SmpB-SsrA system is important for maintaining cellular homeostasis and for survival of bacteria under adverse conditions. Unfortunately, there are only a few studies examining the contribution of this system to bacterial pathogenesis. Previous reports showed that the SmpB-SsrA system plays a critical role in Salmonella pathogenesis through controlling the expression of virulence factors and improving the ability of this organism to survive within macrophages (6, 26). More recently we showed that the smpB-ssrA mutant of Yersinia pseudotuberculosis was avirulent in a mouse infection model (34). Based on this evidence, we investigated the importance of smpB-ssrA in Y. pestis pathogenesis and the possibility of using its mutants as a live cell-based plague vaccine. Our results show that the smpB-ssrA mutant of Y. pestis is severely attenuated in a mouse model of infection. Most importantly, mice vaccinated with this mutant are protected against pulmonary Y. pestis infection.     

Targeting of LcrV virulence protein from Yersinia pestis to dendritic cells protects mice against pneumonic plague

To help design needed new vaccines for pneumonic plague, we targeted the Yersinia pestis LcrV protein directly to CD8α⁺ DEC‐205⁺ or CD8α^? DCIR2⁺ DC along with a clinically feasible adjuvant, poly IC. By studying Y. pestis in mice, we could evaluate the capacity of this targeting approach to protect against a human pathogen. The DEC‐targeted LcrV induced polarized Th1 immunity, whereas DCIR2‐targeted LcrV induced fewer CD4⁺ T cells secreting IFN‐γ, but higher IL‐4, IL‐5, IL‐10, and IL‐13 production. DCIR‐2 targeting elicited higher anti‐LcrV Ab titers than DEC targeting, which were comparable to a protein vaccine given in alhydrogel adjuvant, but the latter did not induce detectable T‐cell immunity. When DEC‐ and DCIR2‐targeted and F1‐V+ alhydrogel‐vaccinated mice were challenged 6 wk after vaccination with the virulent CO92 Y. pestis, the protection level and Ab titers induced by DCIR2 targeting were similar to those induced by F1‐V protein with alhydrogel vaccination. Therefore, LcrV targeting to DC elicits combined humoral and cellular immunity, and for the first time with this approach, also induces protection in a mouse model for a human pathogen.     

A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity.     

Plague in Guinea pigs and its prevention by subunit vaccines

Human pneumonic plague is a devastating and transmissible disease for which a Food and Drug Administration-approved vaccine is not available. Suitable animal models may be adopted as a surrogate for human plague to fulfill regulatory requirements for vaccine efficacy testing. To develop an alternative to pneumonic plague in nonhuman primates, we explored guinea pigs as a model system. On intranasal instillation of a fully virulent strain, Yersinia pestis CO92, guinea pigs developed lethal lung infections with hemorrhagic necrosis, massive bacterial replication in the respiratory system, and blood-borne dissemination to other organ systems. Expression of the Y. pestis F1 capsule was not required for the development of pulmonary infection; however, the capsule seemed to be important for the establishment of bubonic plague. The mean lethal dose (MLD) for pneumonic plague in guinea pigs was estimated to be 1000 colony-forming units. Immunization of guinea pigs with the recombinant forms of LcrV, a protein that resides at the tip of Yersinia type III secretion needles, or F1 capsule generated robust humoral immune responses. Whereas LcrV immunization resulted in partial protection against pneumonic plague challenge with 250 MLD Y. pestis CO92, immunization with recombinant F1 did not. rV10, a vaccine variant lacking LcrV residues 271-300, elicited protection against pneumonic plague, which seemed to be based on conformational antibodies directed against LcrV.     

Neonatal mucosal immunization with a non-living,non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection

Safe and effective immunization of newborns and infants can significantly reduce childhood mortality, yet conventional vaccines have been largely unsuccessful in stimulating the neonatal immune system. We explored the capacity of a novel mucosal antigen delivery system consisting of non-living, non-genetically modified Lactococcus lactis particles, designated as Gram-positive enhancer matrix (GEM), to induce immune responses in the neonatal setting. Yersinia pestis LcrV, used as model protective antigen, was displayed on the GEM particles. Newborn mice immunized intranasally with GEM-LcrV developed LcrV-specific antibodies, Th1-type cell-mediated immunity, and were protected against lethal Y. pestis (plague) infection. The GEM particles activated and enhanced the maturation of neonatal dendritic cells (DCs) both in vivo and in vitro. These DCs showed increased capacities for secretion of proinflammatory and Th1-cell polarizing cytokines, antigen presentation and stimulation of CD4⁺ and CD8⁺ T cells. These data show that mucosal immunization with L. lactis GEM particles carrying vaccine antigens represents a promising approach to prevent infectious diseases early in life.     

Protective Immunity in Mice Achieved with Dry Powder Formulation and Alternative Delivery of Plague F1-V Vaccine

The potential use of Yersinia pestis as a bioterror agent is a great concern. Development of a stable powder vaccine against Y. pestis and administration of the vaccine by minimally invasive methods could provide an alternative to the traditional liquid formulation and intramuscular injection. We evaluated a spray-freeze-dried powder vaccine containing a recombinant F1-V fusion protein of Y. pestis for vaccination against plaque in a mouse model. Mice were immunized with reconstituted spray-freeze-dried F1-V powder via intramuscular injection, microneedle-based intradermal delivery, or noninvasive intranasal administration. By intramuscular injection, the reconstituted powder induced serum antibody responses and provided protection against lethal subcutaneous challenge with 1,000 50% lethal doses of Y. pestis at levels equivalent to those elicited by unprocessed liquid formulations (70 to 90% protection). The feasibility of intradermal and intranasal delivery of reconstituted powder F1-V vaccine was also demonstrated. Overall, microneedle-based intradermal delivery was shown to be similar in efficacy to intramuscular injection, while intranasal administration required an extra dose of vaccine to achieve similar protection. In addition, the results suggest that seroconversion against F1 may be a better predictor of protection against Y. pestis challenge than seroconversion against either F1-V or V. In summary, we demonstrate the preclinical feasibility of using a reconstituted powder F1-V formulation and microneedle-based intradermal delivery to provide protective immunity against plague in a mouse model. Intranasal delivery, while feasible, was less effective than injection in this study. The potential use of these alternative delivery methods and a powder vaccine formulation may result in substantial health and economic benefits.Plague caused by gram-negative Yersinia pestis is one of the most deadly infectious diseases of animals and humans. Outbreaks of plague have caused the deaths of millions of people throughout human history. The pneumonic form of plague is the most dangerous, due to rapid onset and progression and the aerosol spread of the disease. Without proper early treatment, pneumonic plague can lead to mortality in close to 100% of cases (36).Throughout recorded human history, there were three plague pandemics that caused countless human deaths (20). The use of plague as a weapon also has a long history. Starting from the 12th century, there were many cases of using the bodies of plague victims to defeat enemies during wars. Most recently, during World War II, the Japanese army dropped Y. pestis-infected fleas over populated areas of China, causing plague outbreaks (20).Plague has been classified by the U.S. Centers for Disease Control and Prevention as a “category A” agent due to its potential threat to national security. Currently, however, there is no commercially available vaccine against plague approved for human use in the United States. The original plague vaccine licensed for use in the United States was a killed whole-cell bacterial vaccine. It provided some protection against bubonic plague, but not against aerosol exposure to Y. pestis (6, 10, 33, 40). Manufacture of the vaccine ceased in 1998 due to multiple side effects and short-term effectiveness.The potential use of Y. pestis as a bioweapon, combined with the threat of antibiotic-resistant plague (13), makes the development of a safe and effective human plague vaccine a high priority. Conventional vaccines are formulated as liquids, which generally require refrigeration for storage and distribution. In recent years, there has been growing interest in powder formulations for extended storage stability and increased shelf life (5, 12, 19). We previously reported intranasal (i.n.) delivery of dry powder influenza vaccine in a rat model (17). The influenza vaccine powder formulation was shown to be more stable than the liquid vaccine and to induce increased systemic and nasal mucosal immune responses. We also reported that rabbits immunized nasally with a spray-freeze-dried (SFD) anthrax recombinant protective antigen (rPA) powder vaccine formulation were completely protected against lethal inhalational anthrax, while a liquid preparation of the same vaccine provided only 63 to 67% protection by the nasal route (18, 28).The most common vaccine delivery route is intramuscular (i.m.) injection. Alternative delivery methods, such as the intradermal (i.d.) and i.n. routes, have attracted attention recently. The skin is a favorable site for vaccine delivery due to a rich population of antigen-presenting cells. I.d. delivery of vaccines has been shown to provide dose-sparing effects for rabies, hepatitis B, influenza, and rPA vaccines (1, 7, 8, 24, 28, 37). I.n. delivery has been shown to induce mucosal immunity (17, 31). In addition, it offers the advantages of noninvasive delivery and ease of use, reducing the need for highly trained health personnel to administer the vaccine.In the current study, we evaluated the immunogenicity and protective efficacy of an SFD powder vaccine formulation of a recombinant F1-V fusion protein of Y. pestis in a mouse model. The recombinant fusion protein was composed of the F1 subunit, a capsule protein of Y. pestis, and the low-calcium-response V subunit (LcrV), or V antigen. LcrV caps the tips of the injectisome needles of the type III secretion system (30).     

Vaccination with live Yersinia pestis primes CD4 and CD8 T cells that synergistically protect against lethal pulmonary Y. pestis infection

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 na?ve 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.     

Dual-Function Antibodies to Yersinia pestis LcrV Required for Pulmonary Clearance of Plague

Yersinia pestis causes pneumonic plague, a necrotic pneumonia that rapidly progresses to death without early treatment. Antibodies to the protective antigen LcrV are thought to neutralize its essential function in the type III secretion system (TTSS) and by themselves are capable of inducing immunity to plague in mouse models. To develop multivalent LcrV antibodies as a therapeutic treatment option, we screened for monoclonal antibodies (MAbs) to LcrV that could prevent its function in the TTSS. Although we were able to identify single and combination MAbs that provided the high-level inhibition of the TTSS, these did not promote phagocytosis in vitro and were only weakly protective in a mouse pneumonic plague model. Only one MAb, BA5, was able to protect mice from pneumonic plague. In vitro, MAb BA5 blocked the TTSS with efficiency equal to or even less than that of other MAbs as single agents or as combinations, but its activity led to increased phagocytic uptake. Polyclonal anti-LcrV was superior to BA5 in promoting phagocytosis and also was more efficient in protecting mice from pneumonic plague. Taken together, the data support a hypothesis whereby the pulmonary clearance of Y. pestis by antibodies requires both the neutralization of the TTSS and the simultaneous stimulation of innate signaling pathways used by phagocytic cells to destroy pathogens.Yersinia pestis, the etiologic agent of bubonic, pneumonic, and septicemic plague, has been responsible for more human death than any other bacterial pathogen (42). Fortunately, naturally occurring cases of plague in humans now are uncommon, largely due to advances in basic sanitation and public awareness of infectious disease (32). Nevertheless, the disease remains endemic in many areas of the world, and periodic human bubonic and, to a lesser extent, pneumonic plague cases appear each year. Yersinia pestis is believed to have evolved recently from Yersinia pseudotuberculosis, acquiring flea transmission and respiratory invasion properties through mobile genetic elements (1, 9). The flea transmission cycle provides an opportunity for further evolution, because the bacteria reside in the nonsterile environment of the flea gut, where the formation of a biofilm provides an opportunity for horizontal gene exchange with other microbes (30). Multidrug-resistant Y. pestis isolates have been recovered from human plague patients, suggesting that the bacteria do indeed continue to evolve mechanisms of survival in the mammalian host (22, 25, 54). For these reasons, as well as for its potential use as a biological weapon, Y. pestis continues to be a significant public health concern and is a priority pathogen for the development of new vaccines and alternative therapeutics (32, 43).There currently are no plague vaccines that are licensed for human use in the United States. The licensing of current candidates is likely to fall under the U.S. Food and Drug Administration''s Animal Rule for the demonstration of efficacy and potency due to a lack of naturally occurring human plague cases (19). Thus, efficacy trials and the evaluation of vaccine potency in humans will be dependent on our ability to understand the molecular mechanism of protection. Current subunit vaccine candidates are formulated from two protective antigens, Fraction 1 (F1) and LcrV, which are undergoing extensive testing to satisfy the Animal Rule requirements (2, 5, 13, 26, 55, 57-59). Both antigens elicit a neutralizing antibody response that can be translated to passive antibody or even gene therapies (2, 4, 13, 28, 37, 48). These protective antibodies act directly on the bacteria and alter its interactions with innate immune cells such that the host clears the infection. T-cell responses also are believed to play an important role in host defense against Yersinia pestis (40, 41).CaF1, or F1, is an abundant cell surface antigen of the type I pilin family that forms a capsule-like structure on Y. pestis at 37°C (8). Although F1 appears to be antiphagocytic, it is not essential for virulence and thus would not contribute to immunity against Y. pestis mutant caF1 (18, 21). In contrast, LcrV is essential for all forms of plague due to its role in the type III secretion system (TTSS) (12, 45, 47). LcrV is positioned on the surface of bacteria at 37°C, where it mediates the translocation of anti-host factors, collectively known as Yersinia outer proteins (Yops), whose antiphagocytic, cytolytic, and proapoptotic activities allow Yersinia to avoid being killed by the host''s immune system (38, 46). Polyclonal antibodies to recombinant LcrV (α-LcrV) can bind to this needle tip and lead to the inhibition of the TTSS and the phagocytosis of the bacteria (14, 24, 53). However, it remains controversial whether the direct inhibition of the TTSS by α-LcrV leads to phagocytosis or if the direct promotion of phagocytosis leads to the inhibition of the TTSS because it cannot function intracellularly (59, 60). Three monoclonal antibodies (MAbs) have been independently cloned that can protect mice from bubonic and pneumonic plague (2, 27, 48). Although it is unclear whether each of these targets the same epitope, deletion studies of LcrV antigen suggest multiple protective epitopes exist (13, 39, 44, 51).We were interested in developing antibody therapeutics and maximizing the potency of anti-LcrV therapy. In this work, we investigated the mechanism of protection from pneumonic plague to determine if the multivalent occupancy of antibody to LcrV improved protection. We found that antibodies that promoted phagocytosis directly were more potent at neutralizing pneumonic plague, although the inhibition of the TTSS alone led to partial protection. Only a single LcrV epitope led to antibodies that by themselves promoted uptake, while the multivalent occupation of antigen with MAbs did not increase either phagocytosis or protection. These data provide new insight into the mechanism of LcrV and support the use of assays that measure the phagocytic uptake of Y. pestis as correlates of immunity for the evaluation of plague vaccines.     

Yersinia pestis caf1 variants and the limits of plague vaccine protection

Yersinia pestis, the highly virulent agent of plague, is a biological weapon. Strategies that prevent plague have been sought for centuries, and immunization with live, attenuated (nonpigmented) strains or subunit vaccines with F1 (Caf1) antigen is considered effective. We show here that immunization with live, attenuated strains generates plague-protective immunity and humoral immune responses against F1 pilus antigen and LcrV. Y. pestis variants lacking caf1 (F1 pili) are not only fully virulent in animal models of bubonic and pneumonic plague but also break through immune responses generated with live, attenuated strains or F1 subunit vaccines. In contrast, immunization with purified LcrV, a protein at the tip of type III needles, generates protective immunity against the wild-type and the fully virulent caf1 mutant strain, in agreement with the notion that LcrV can elicit vaccine protection against both types of virulent plague strains.     

Exogenous Yersinia pestis quorum sensing molecules N-octanoyl-homoserine lactone and N-(3-oxooctanoyl)-homoserine lactone regulate the LcrV virulence factor

LcrV is a key Yersinia pestis antigen, immune regulator, and component of the type III secretion system (T3SS). Researchers have shown that N-acyl-homoserine lactones (AHLs) can down-regulate the expression of the LcrV homolog, PcrV, in Pseudomonas aeruginosa. Using ELISA, western blot, DNA microarray analysis, and real time PCR we demonstrate that the addition of AHL molecules N-octanoyl-homoserine lactone (C8) or N-(3-oxooctanoyl)-homoserine lactone (oxo-C8) to Y. pestis cultures down-regulates LcrV protein expression. DNA microarray analysis shows 10 additional T3SS genes are consistently down-regulated by C8 or oxo-C8. This is the first report demonstrating that AHLs regulate Y. pestis virulence factor expression.     

Impact of the Pla Protease Substrate α2-Antiplasmin on the Progression of Primary Pneumonic Plague

Many pathogens usurp the host hemostatic system during infection to promote pathogenesis. Yersinia pestis, the causative agent of plague, expresses the plasminogen activator protease Pla, which has been shown in vitro to target and cleave multiple proteins within the fibrinolytic pathway, including the plasmin inhibitor α2-antiplasmin (A2AP). It is not known, however, if Pla inactivates A2AP in vivo; the role of A2AP during respiratory Y. pestis infection is not known either. Here, we show that Y. pestis does not appreciably cleave A2AP in a Pla-dependent manner in the lungs during experimental pneumonic plague. Furthermore, following intranasal infection with Y. pestis, A2AP-deficient mice exhibit no difference in survival time, bacterial burden in the lungs, or dissemination from wild-type mice. Instead, we found that in the absence of Pla, A2AP contributes to the control of the pulmonary inflammatory response during infection by reducing neutrophil recruitment and cytokine production, resulting in altered immunopathology of the lungs compared to A2AP-deficient mice. Thus, our data demonstrate that A2AP is not significantly affected by the Pla protease during pneumonic plague, and although A2AP participates in immune modulation in the lungs, it has limited impact on the course or ultimate outcome of the infection.     

Biosafety Level 2 Model of Pneumonic Plague and Protection Studies with F1 and Psa

Attenuated Yersinia pestis pgm strains, such as KIM5, lack the siderophore yersiniabactin. Strain KIM5 does not induce significant pneumonia when delivered intranasally. In this study, mice were found to develop pneumonia after intranasal challenge with strain KIM5 when they were injected intraperitoneally with iron dextran, though not with iron sulfate. KIM5-infected mice treated daily with 4 mg iron dextran died in 3 days with severe pneumonia. Pneumonia was less severe if 4 mg iron dextran was administered only once before infection. The best-studied experimental vaccine against plague currently consists of the Yersinia pestis capsular antigen F1 and the type 3 secreted protein LcrV. The F1 antigen was shown to be protective against KIM5 infections in mice administered iron dextran doses leading to light or severe pneumonia, supporting the use of an iron dextran-treated model of pneumonic plague. Since F1 has been reported to be incompletely protective in some primates, and bacterial isolates lacking F1 are still virulent, there has been considerable interest in identifying additional protective subunit immunogens. Here we showed that the highly conserved Psa fimbriae of Y. pestis (also called pH 6 antigen) are expressed in murine organs after infection through the respiratory tract. Studies with iron dextran-treated mice showed that vaccination with the Psa fimbrial protein together with an adjuvant afforded incomplete but significant protection in the mouse model described. Therefore, further investigations to fully characterize the protective properties of the Psa fimbriae are warranted.Yersinia pestis, a Gram-negative bacterium belonging to the family Enterobacteriaceae, is the causative agent of plague, a disease that affects a variety of mammalian hosts and that can be transmitted by various arthropod vectors. Humans are susceptible to Y. pestis, whether transmitted by aerosol or by infected fleas, causing the highly lethal pneumonic plague or bubonic plague, respectively (53). Aerosol infections by Y. pestis remain a concern for public health (12). The only currently available tools for combating Y. pestis in the United States are antibiotics, since manufacture of the previously licensed formalin-killed Y. pestis vaccine that was used by the U.S. military during the Vietnam War (41) stopped in 1998. Although effective against bubonic plague, this vaccine was not efficiently protective against pneumonic plague and caused significant side effects (11). The emergence of multiple antimicrobial resistance in Y. pestis (72) and the lack of a safe and effective vaccine have been the impetus for the search for new inhibitory drugs as well as a better vaccine (48, 49).Y. pestis has been classified as a Category A select agent (8) requiring special precautions, particularly because of the risk of pneumonic plague due to bacterial aerosolization. One of the major impediments to studying the biology and pathogenesis of Y. pestis is that many laboratories do not have access to the required biosafety level 3 (BSL-3) facilities and therefore must work with attenuated strains that were excluded from the CDC select-agents list. Such strains lack either the pgm locus (e.g., KIM5) or the virulence plasmid pCD1 (51). A major virulence component of the pgm locus is the yersiniabactin (Ybt siderophore)-dependent iron transport system, which is essential for plague infection from peripheral sites (52). The pCD1 plasmid encodes a type III secretion system (T3SS, or Yops regulon) that, upon contact with host cells (or in low calcium concentrations in vitro), produces an elaborate injection machinery used to transfer a set of effector proteins with potent antiphagocytic and/or anti-inflammatory effects (2, 67) directly to the cytosol of the eukaryotic cell. Mutants lacking pgm or pCD1 are safer to manipulate, allowing experimental studies to be carried out in BSL-2 laboratories. However, the disease progression elicited by these strains does not recapitulate that observed with fully virulent strains (44), and in comparison to the wild-type strain, these strains are highly attenuated by any natural route of infection. Specifically, the Δpgm strain KIM5 is attenuated when given subcutaneously (s.c.) (52). Similarly, another Δpgm derivative of the virulent strain KIM, strain D27 (KIM D27), was unable to grow to high numbers in the lungs of intranasally infected mice, suggesting that bacterial growth was contained by the host''s innate immune system. Moreover, these mice did not develop significant pneumonia, as they would if infected with wild-type Y. pestis strains (33). However, strain KIM D27 was still able to spread and grow in spleens and livers. This suggested that the iron uptake system of the pgm locus was needed to counteract iron restriction in the lungs. Interestingly, the results of earlier studies suggested that parenteral administration of iron sulfate to mice could bypass the need for yersiniabactin, rendering a parenterally administered Y. pestis Δpgm mutant fully virulent (10). However, a recent study showed that the use of the same approach to induce pneumonic plague after intranasal (i.n.) administration of pgm strain KIM D27 was not successful (33). This study also highlighted the toxicity of ferrous chloride, limiting administration doses to 0.5 mg per mouse. The latter result was consistent with the early toxicity data for inorganic iron at 30 to 60 mg/kg of body weight (26).The toxicity problem of inorganic iron and the need for iron treatments for various medical conditions led to the development of less-toxic colloids consisting of various carbohydrates with ferric (oxy)hydroxide (16). In agreement with the results of earlier studies with inorganic iron, intraperitoneal (i.p.) administration of colloidal iron to mice subsequently infected subcutaneously with a Y. pestis pgm strain resulted in death, with histological lesions in the livers and spleens resembling those resulting from infection with a virulent strain (68). A variety of more-recent models of bacterial infections were developed by taking advantage of the extremely low toxicity of the colloid iron dextran, administrations of which in the milligram range were shown to be nontoxic (29, 62, 75). Based on the success of these models, we undertook experiments to determine whether the administration of high doses of iron dextran would permit Y. pestis to grow in the lungs of i.n. infected mice and cause local inflammation. Here we describe the development of a BSL-2 pneumonic plague model in mice based on the administration of iron dextran. In contrast to the negative results obtained with iron chloride, the use of iron dextran has allowed us to bypass toxicity problems and to take advantage of its favorable pharmacokinetic properties, such as slower clearance and a longer half-life for iron than those with other colloidal or inorganic iron preparations (16).Our model was tested by confirming the expected protective property of F1 in pneumonic plague. We also used the model to evaluate the potential protective property of the Psa fimbriae (pH 6 antigen). Both F1 and Psa are surface structures that share the structural properties of homopolymeric fimbriae, each formed by a protein subunit that requires its own chaperone and usher proteins for export. Structural studies have classified F1 and Psa within a family of fimbrial polyadhesins assembled by FGL chaperones (for their long F1 and G1 β-strands) (76). The F1 capsular antigen is an efficient immunogen that is protective by itself in a murine model of pneumonic plague with F1⁺ strains (4). The protective property of the F1 immunogen can be attributed in great part to its polymeric structure and in vivo surface exposure. Similarly, mixtures of fimbriae and surface-exposed adhesins of other pathogens have been used successfully as commercial vaccine components for both humans and animals (17, 43, 54). Surface-expressed antigens such as F1 induce opsonizing and/or antivirulence antibodies, and such antibodies by themselves are protective (28). A role for opsonization by antibodies directed toward Y. pestis surface antigens is suggested by studies that showed that neutrophil depletion (15) or inhibition of cytokines that boost the antimicrobial activity of macrophages (18, 50) interfered with antibody-mediated protection. Properties of Psa similar to those of F1, such as its polymeric structure and bacterial surface location, make it an attractive vaccine candidate. Therefore, we evaluated Psa for its in vivo expression, immunogenicity, and protective properties by using the pneumonic plague model that we developed.     

Fine-Tuning Synthesis of Yersinia pestis LcrV from Runaway-Like Replication Balanced-Lethal Plasmid in a Salmonella enterica Serovar Typhimurium Vaccine Induces Protection against a Lethal Y. pestis Challenge in Mice

A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal ΔasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain χ9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P_cro) (P_R), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P_BAD c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of β-lactamase, and cloned into pYA4534 under the control of the P_trc promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain χ9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.Live, attenuated bacteria have been developed to generate safe and immunogenic vaccine strains (50). Attenuated Salmonella enterica has been used as both a homologous vaccine and a delivery system for recombinant heterologous antigens from bacterial, parasitic, viral, and tumor sources (8, 40). The oral administration of Salmonella allows the infection of Peyer''s patches via the M cells and colonization of the mesenteric lymph nodes, liver, and spleen, generating a range of humoral and cellular immune responses against Salmonella and the heterologous antigens (8) at local and distal sites such as the mucosa. Because most systems for the expression of heterologous antigenic proteins in Salmonella use plasmids, several approaches have been developed for the antibiotic-free maintenance of plasmid vectors (29, 48). However, a number of factors may affect the immune response to protective antigens, such as the ability of the vaccine strain to invade and colonize the host and the stability of the plasmid expression system. High levels of bacterial protein synthesis specified by multiple-copy plasmids often result in either the rapid loss of the foreign plasmid or a reduction in bacterial growth and the ability to colonize lymphoid tissues due to the demand of the extrametabolic burden. Both of these factors result in a reduction of immunogenicity. The insertion of genes into the bacterial chromosome by homologous recombination can achieve a high degree of stability, but this approach sometimes limits the level of protein synthesis due to the single gene copy and, thus, may lessen the production of a protective immune response with the live vaccine (29).To overcome some of these problems, we have constructed a balanced-lethal vaccine vector with its copy number regulated by arabinose (pYA4534) that switches to runaway-like high-copy-number replication regulating the delivery and dosage of heterologous antigens. In previous work, the switch from a low to a high copy number of the plasmid was mediated by a temperature change from 30°C to higher than 35°C and is called uncontrolled replication or runaway replication of the plasmid in Escherichia coli (62).Yersinia pestis is a Gram-negative bacterium that causes plague in humans and is transmitted from rodents to humans by fleas (26, 51). Y. pestis infections present three different clinical forms: bubonic, pneumonic, or septicemic (59). Widespread aerosol dissemination of the bacterium combined with high mortality rates make Y. pestis a deadly pathogen (31). LcrV is a multifunctional protein that forms part of a type III secretion system (T3SS) encoded on Y. pestis 70-kb virulence plasmid pCD1 (16, 52). LcrV along with LcrG helps regulate the expression of Yersinia outer proteins (YOPs) that are injected into the cytosol of the host cell, where they interfere with the cellular signaling involved in phagocytosis and inhibit proinflammatory cytokine production (28, 43, 47). Experimental evidence indicates that antibody responses to LcrV offer protection against plague. Thus, the passive transfer of LcrV monoclonal antibodies (MAbs) or polyclonal-specific serum to LcrV protects animals against bubonic and pneumonic plague (25, 46). Antibodies against LcrV apparently block the translocation of effector YOPs, allowing the phagocytosis of Y. pestis bacilli by macrophages, but the exact mechanism of this protection remains to be determined (19).In addition to the direct role of LcrV in the formation of the T3SS needle, LcrV has an immunomodulatory function mediated by interleukin-10 (IL-10) induction, which blocks the host protective inflammatory responses and suppresses the proinflammatory cytokines (7). Partial deletions of LcrV and the use of synthetic peptides allowed the identification of two LcrV regions involved in the production of IL-10, which are located from amino acid residues 37 to 57 and from amino acid residues 271 to 285 (34, 49). The induction of IL-10 by LcrV is through the interaction of Toll-like receptor 2 (TLR-2) as well as TLR-6 and cluster of differentiation 14 (CD14) (1, 15, 58). Immunization with full-length LcrV elicited protective immunity (41), but truncated LcrV forms were also able to elicit an immune response that was protective against a lethal challenge with Y. pestis. These variants included rV10 (lacking amino acid residues 271 to 300) (49), the major protective LcrV region (amino acid residues 135 to 275) (25), LcrV196 (amino acid residues 131 to 326) (5), and a small fragment of LcrV (amino acids 135 to 262) (64).In this work, we describe the construction of pYA4534, a balanced-lethal plasmid expression system containing an arabinose-regulated genetic switch to shift to runaway-like high-copy-number replication in vivo for the regulated delayed dosage of heterologous antigens. The derivative pYA4535, encoding the T2SS β-lactamase N- and C-terminal domains for the export of the lcrV196-encoded fused antigen, was used to transform S. enterica serovar Typhimurium strain χ9447, a new generation of live recombinant attenuated Salmonella vaccines (RASVs) that is phenotypically similar to the wild type at the time of oral vaccination but displays a regulated delayed in vivo attenuation (14), a regulated delayed in vivo synthesis of recombinant antigen (66), and regulated delayed in vivo lysis to release a bolus of protective antigen and confers complete biological containment (35) after host tissue colonization. These RASV strains are able to colonize and persist in the lymphoid tissue without causing disease symptoms, giving an advantageous alternative when carrying heterologous antigens that induce higher protective mucosal and systemic immunity responses. The immune responses of mice immunized orally with this live RASV strain synthesizing an optimized LcrV protein were evaluated for protection against a lethal challenge with virulent Y. pestis CO92 (4). Thus, we offer an alternative for the development of vaccines against clinical forms of plague.