Abstract Introduction Yersinia pestis (Yp), a facultative anaerobic gram-negative bacillus, causes plague, which if untreated has a case-fatality rate approaching 100% in primary pneumonic form. Yp is a known biothreat agent that can be aerosolized to potentially cause pneumonic plague outbreaks, and there is no licensed vaccine. Methods Here we used a pneumonic plague model in which BALB/c mice were exposed to aerosolized Yp strain CO92 to compare DNA vaccines encoding Yp antigens LcrV and F1 with established adjuvanted subunit vaccines based on LcrV and F1 proteins. DNA was delivered using a needle-free PharmaJet Tropis device recently modified for use in small animals, with no additional antigens or adjuvants. Results After two vaccine doses, mice vaccinated with F1 and/or LcrV DNA had cellular immunity comparable to the most effective subunit vaccines, as measured by cytokine secretion and counts of IFN-γ-producing splenocytes after ex vivo restimulation. LcrV DNA vaccination generated high anti-LcrV serum antibody titers, was effective at blocking bacterial replication, and conferred 60% survival in this model where all unvaccinated animals died within 4 days of challenge. The F1 DNA construct was not protective despite inducing T-cell immunity and was redesigned using information from the literature to address this deficiency. Conclusion These results are promising for the needle-free DNA/jet vaccine platform against bacterial pathogens, and studies of LcrV DNA combined with redesigned F1 DNA are in progress. Funding Source DTRA-JSTO Topic Categories Vaccines and Immunotherapy (VAC)

Abstract Introduction Buprenorphine is a potent partial opioid used to manage pain and opioid dependence. The buprenorphine extended-release formulation, Ethiqa XR (Ethiqa), has been FDA-indexed for mice and other laboratory species to relieve pain, however, several reports indicate that Ethiqa can alter the inflammatory response in various in vivo models. Therefore, it is necessary to understand the effect of Ethiqa on disease pathogenesis prior to its use during animal studies. Methods Here, we evaluated the effects of Ethiqa treatment prior to and 48h after bacterial challenge of C57BL/6 and BALB/c mice with Burkholderia pseudomallei and Yersinia pestis. Results Both mouse strains challenged with aerosolized Burkholderia pseudomallei K96243 demonstrated increased bacterial dissemination to the spleen when treated with Ethiqa. Consistently, splenocytes had elevated pro-inflammatory cytokines such as IFN-gamma, IL-6, IL-22, TNF-alpha, among others, as well as elevated levels of iNOS. The C57BL/6 mice also displayed elevated IFN-gamma and IP-10/CXCL10 in lung and brain homogenates. In contrast, no profound differences were observed in either mouse strain treated with Ethiqa and challenged with Yersinia pestis CO92. In the absence of challenge, treatment with Ethiqa did not change the cytokine responses in the lung, brain, or spleen of BALB/c mice but increased the concentration of IL-6 and TNF-alpha in the lungs of C57BL/6 mice. Conclusion Taken together, these results suggest the effects of Ethiqa may be model-specific and introduce confounding effects that would impact dependent variables. Funding Source DTRA-JSTO Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)

Abstract Introduction Yersinia pestis (Yp) and Burkholderia pseudomallei (Bp), the causative agents of plague and melioidosis respectively, are both responsible for extremely high lethality. There are no FDA-approved vaccines against these pathogens, and treatments are challenging due to antibiotic resistance. Methods Here we developed a multi-pathogen vaccine against Yp and Bp. Mice were vaccinated with a nanolipoprotein (NLP) formulated with Yp Fraction 1 protein (F1), Yp low calcium response V protein (V), Bp hemolysin-coregulated protein 1 (Hcp1) as well as Bp capsular polysaccharide (CPS) admixed with CpG and aluminum hydroxide wet gel (AlOH), following a two-dose vaccine schedule. We used a model of pneumonic plague and melioidosis in which C57BL/6 mice were initially aerosol challenged with Yp, and following convalescence mice were then challenged with Bp. Results The multi-pathogen vaccine conferred 100% and 60% protection following Yp and Bp challenge, respectively. Anti-F1, -V, and -Hcp1 IgG titers in serum reached similar levels between all vaccine groups and appeared to diminish at a similar rate. The magnitude of the IFN-gamma recall response of splenocytes was less pronounced in the multi-pathogen vaccine, although it conferred similar protection as current benchmark vaccines. Conclusion The multi-pathogen vaccine was comparably protective to benchmark vaccines but with the advantage of reduced number of injections and dose sparing. These data show that the NLP platform is amenable to multi-pathogen formulations and a single vaccine regimen can protect from both pneumonic plague and melioidosis. Funding Source DTRA-JSTO Topic Categories Vaccines and Immunotherapy (VAC)

Abstract Introduction Plague, caused by the bacterium Yersinia pestis, can present as several forms including bubonic, septicemic, pneumonic, and meningitis. Plague meningitis is considered rare and has been estimated to occur in 6-11% of documented cases, mostly observed in pediatric patients 15 years old or younger. Furthermore, most cases are tied to inadequate or no antibiotic treatment of bubonic cases. To date, there have been few reports describing neurologic plague in the mouse model of pneumonic plague. Methods We used the BALB/c mouse model of exposure to aerosolized Y. pestis to characterize the pathogenic effects in the CNS resulting from pneumonic plague. Here we use a multifaceted approach analyzing tissue homogenates, tissue slides, transcriptomic, and cytokine data of mice exposed to aerosolized Y. pestis CO92, collected at 24-hour intervals for three days. Results We find that Y. pestis is present in brain homogenates, but histology indicates that it is bound to the capillaries inside the cerebellum, cerebrum, and nasal turbinates. However, transcriptomic data targeting mouse neuroinflammatory responses indicates dysregulation of several gene pathways including those associated with CNS cells such as astrocytes, oligodendrocytes, and microglia. Furthermore, there is a cytokine response to pneumonic plague in brain tissues. Conclusion Although Y. pestis does not breach the capillaries resulting in meningitis in our model, we find evidence of a neuroinflammatory response within the CNS of mice. Funding Source DTRA-JSTO Topic Categories Neuroimmunology (NEUR)

Abstract Introduction Melioidosis, an infectious disease caused by Burkholderia pseudomallei (Bp), is difficult to treat due to intrinsic antibiotic resistance, latency with subsequent recrudescence and a diverse non-specific symptomatology. A vaccine is needed to effectively control this disease burden around the globe. Here we tested the efficacy of a novel Bp vaccine containing hemolysin-coregulated protein (Hcp1), capsular polysaccharide (CPS)-CRM197 conjugate, and CpG using the Atomic Layering Thermostable Antigen and Adjuvant (ALTA®) platform. Methods C57BL/6 mice were vaccinated and the protective efficacy as well as immunological profiles were evaluated pre- and post-aerosol challenge with B. pseudomallei K96243 strain. Results Each tested dose of Bp ALTA® formulation demonstrated protective efficacy (20-50%) over a non-vaccinated control. Mechanistically, the Bp ALTA® formulations increased Hcp1-directed IFN-gamma recall in splenocytes, and produced robust antibody response against the principal dominant antigen, CPS. High levels of anti-CPS antibodies were associated with significant decrease in bacterial burden in the lung. Conclusion The ALTA® platform, which has previously been shown to provide antigen thermostability, was utilized in this study for Bp vaccination achieving protective efficacy from a single-shot administration. Studies are ongoing to optimize the immunogenic potential and protective capacity of these novel formulations. Funding Source DTRA-JSTO Topic Categories Vaccines and Immunotherapy (VAC)

Abstract Introduction Yersinia pestis (Yp) is a gram-negative bacillus that causes plague in humans and animals and is endemic in Africa, Asia, and the Americas. When aerosolized, bacterium can cause an acute, often fatal disease that requires antibiotic intervention within 24hr of exposure. Antibodies have been previously raised against two protective antigens, F1 and LcrV, unfortunately Yp is able to circumvent these countermeasures. Methods To expand the breadth of protective targets we generated cow anti-sera against four novel antigens with potential induction of bovine exclusive ultralong CDR H3 antibodies. Cows were administered six doses of Pla, LptED, Ail and BamA, 3-4 weeks apart and antibody titers were evaluated throughout the duration of the study. Results Functional activity of anti-sera was evaluated utilizing gentamicin protection assay, by enumeration of internalized Yp by RAW cells in the presence or absence of complement. A prominent anti-LptED antibody response developed early during the course of vaccination, followed by anti-Pla, -Ail and -BamA. The number of RAW cells that internalized viable Yp was considerably lower in the presence of fresh or C8 repleted serum relative to heat inactivated or C8 depleted sera. Conclusion The inhibitory properties of cow anti-sera against four Yp antigens will enable identification of novel protective targets that will be used to produce additional countermeasures, especially if they stem from the ultralong CDR H3 repertoire. Funding Source DTRA-JSTO Topic Categories Vaccines and Immunotherapy (VAC)

Abstract Yersinia pestis (Yp) is the etiological agent of plague, a disease that remains a concern as demonstrated by recent outbreaks in Madagascar. Infection with Yp results in a rapidly progressing illness that can only be treated with antibiotics. Live attenuated or whole-cell inactivated vaccines confer short-lived protection against bubonic but not pneumonic plague. Subunit vaccine formulations may circumvent some of these shortfalls. Here we compare the immunogenicity generated by the most advanced subunit vaccine (rF1V) against a nanolipoprotein particle (NLP)-based vaccine. BALB/c mice were immunized twice, four weeks apart. Four weeks later, splenocytes and sera were collected for immune profiling and mice were aerosol challenged with Yp CO92. Both formulations induced a strong IgG response against F1 and V proteins, along with a robust memory B cell response and cell-mediated response with Th1- and Th2-related cytokines. NLP-based vaccine induced a stronger cytokine response against F1, V, and F1V proteins relative to the rF1V vaccine. As with rF1V, the inclusion of Alhydrogel in NLP vaccine formulations was critical for enhanced immunogenicity and protective efficacy. Addition of Francisella tularensis antigens to the Yp NLP vaccine platform did not impair vaccine efficacy, based on protection and antibody response. The modularity and lipid bilayer structure of NLPs allows for incorporation of many protective antigens with variable ratios against multiple pathogens.

BackgroundYersinia pestis is the etiological agent of plague, which can manifest as bubonic, septicemic, and/or pneumonic disease. Plague is a severe and rapidly progressing illness that can only be successfully treated with antibiotics initiated early after infection. There are no FDA-approved vaccines for plague, and some vaccine candidates may be less effective against pneumonic plague than bubonic plague. Y. pestis is not known to impact males and females differently in mechanisms of pathogenesis or severity of infection. However, one previous study reported sex-biased vaccine effectiveness after intranasal Y. pestis challenge. As part of developing a safe and effective vaccine, it is essential that potential sex differences are characterized. MethodsIn this study we evaluated novel vaccines in male and female BALB/c mice using a heterologous prime-boost approach and monitored survival, bacterial load in organs, and immunological correlates. Our vaccine strategy consisted of two subcutaneous immunizations, followed by challenge with aerosolized virulent nonencapsulated Y. pestis. Mice were immunized with a combination of live Y. pestis pgm- pPst-Δcaf1, live Y. pestis pgm- pPst-Δcaf1/ΔyopD, or recombinant F1-V (rF1-V) combined with adjuvants. ResultsThe most effective vaccine regimen was initial priming with rF1-V, followed by boost with either of the live attenuated strains. However, this and other strategies were more protective in female mice. Males had higher bacterial burden and differing patterns of cytokine expression and serum antibody titers. Male mice did not demonstrate synergy between vaccination and antibiotic treatment as repeatedly observed in female mice.ConclusionsThis study provides new knowledge about heterologous vaccine strategies, sex differences in plague-vaccine efficacy, and the immunological factors that differ between male and female mice.

Abstract Pneumonic plague, caused by the bacterium Yersinia pestis, is a severe and rapidly progressing illness transmitted via aerosol. There are no FDA-approved vaccines, and candidate vaccines may be less effective against pneumonic than bubonic plague. Y. pestis is not known to impact males and females differently in mechanism of pathogenesis or severity. However, a recent study reported sex-biased vaccine effectiveness after intranasal challenge. In this study we observed that when male mice were given a vaccine that is protective in females challenged with aerosolized virulent Y. pestis C12, males were not protected. We then gave male and female BALB/c mice a recently developed heterologous vaccine strategy and monitored survival, bacterial burden (CFU), and immunological factors before and after aerosol challenge. This strategy consisted of two subcutaneous injections: one of Y. pestis live attenuated vaccine (LAV) strain pgm-pPst-Δcaf1 or pgm-pPst-ΔyopD/Δcaf1 and one of rF1-V protein subunit with adjuvants CpG and Alhydrogel. When primed with rF1-V followed by pgm-pPst-Δcaf1 LAV boost, 100% of females and 50% of males survived challenge. This sex difference was observed to a lesser extent with three other vaccines. Males had higher CFU titers and differing patterns of serum antibodies, lung cytokines, and splenic T cells. These data provide new knowledge about sex differences in vaccine efficacy and immunological factors that differ between males and females.

Abstract Yersinia pestis (Yp) is a security concern since it can be aerosolized and cause pneumonic plague, a rapidly progressing illness that is fatal if not treated promptly with antibiotics. There are no FDA-approved vaccines, and candidate vaccines are less effective against pneumonic than bubonic plague. The major protective vaccine antigen has been the F1 capsule protein; protection against nonencapsulated strains has been elusive. We constructed two novel live attenuated vaccine (LAV) strains on the pgm-pPst- background, which is excluded from the CDC select agent list due to established safety. These strains, Δcaf1 and ΔyopD/Δcaf1, lack F1 and may be more effective at inducing immunity against nonencapsulated strains like C12. Female BALB/c mice were immunized with a heterologous prime-boost strategy combining one subcutaneous injection of novel LAV and one of recombinant F1-V (rF1-V) subunit, then challenged with aerosolized virulent Yp C12 and monitored for survival, bacterial load, and immunological factors. Priming with rF1-V and boosting with LAV was more protective than the reverse strategy. Protection was accompanied by high antibody titers against rF1-V and rV. rF1-V prime and LAV boost also led to a higher IgG2a/IgG1 antibody ratio than the reverse, suggesting a balanced Th1/Th2 response. These data support rF1-V prime and pgm-pPst-Δcaf1 LAV boost as a promising strategy for a safe and effective vaccine against both encapsulated and nonencapsulated Yp.

Yersinia pestis, the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y. pestis. We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y. pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 μg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y. pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague.

In the United States in 2021, an outbreak of 4 cases of Burkholderia pseudomallei, the etiologic agent of melioidosis and a Tier One Select Agent (potential for deliberate misuse and subsequent harm), resulted in 2 deaths. The causative strain, B. pseudomallei ATS2021, was unintentionally imported into the United States in an aromatherapy spray manufactured in India. We established that ATS2021 represents a virulent strain of B. pseudomallei capable of robust formation of biofilm at physiologic temperatures that may contribute to virulence. By using mouse melioidosis models, we determined median lethal dose estimates and analyzed the bacteriologic and histopathologic characteristics of the organism, particularly the potential neurologic pathogenesis that is probably associated with the bimABm allele identified in B. pseudomallei strain ATS2021. Our data, combined with previous case reports and the identification of endemic B. pseudomallei strains in Mississippi, support the concept that melioidosis is emerging in the United States.

Yersinia pestis is a gram-negative bacterium that causes plague in animals and humans. Depending on the route of disease transmission, the bacterium can cause an acute, often fatal disease that has a narrow window for treatment with antibiotics. Additionally, antibiotic resistant strains have been identified, emphasizing the need for novel treatments. Antibody therapy is an appealing option that can direct the immune system to clear bacterial infections. Advances in biotechnology have made both engineering and producing antibodies easier and more affordable. In this study, two screening assays were optimized to evaluate the ability of antibodies to promote phagocytosis of Y. pestis by macrophages and to induce a cytokine signature in vitro that may be predictive of protection in vivo. We evaluated a panel of 21 mouse monoclonal antibodies targeting either the anti-phagocytic capsule F1 protein or the LcrV antigen, which is part of the type 3 secretion system that facilitates translocation of virulence factors into the host cell, using two functional assays. Anti-F1 and anti-LcrV monoclonal antibodies both increased bacterial uptake by macrophages, with greater uptake observed in the presence of antibodies that were protective in the mouse pneumonic plague model. In addition, the protective anti-F1 and anti-LcrV antibodies produced unique cytokine signatures that were also associated with in vivo protection. These antibody-dependent characteristics from in vitro functional assays will be useful in down-selecting efficacious novel antibodies that can be used for treatment of plague.

Burkholderia pseudomallei , the causative agent of melioidosis, has two phases of infection. The acute phase occurs shortly after infection and is associated with bacterial sepsis, potentially leading to death, whilst the chronic phase occurs when infection persists for longer periods or is asymptomatic for months or years. BALB/c mice are more susceptible to melioidosis compared to C57BL/6 mice and are routinely models for the acute phase of infection. However, in some instances when medical countermeasures are being evaluated, mice continue to succumb to disease throughout the course of the experimental infection. Whilst B. pseudomallei is not known to be transmitted from mouse-to-mouse, we hypothesized that mice that have recovered from infection after medical countermeasure intervention may become reinfected from chronically infected mice. We tested this hypothesis by cohousing naïve mice with mice exposed to B. pseudomallei by the inhalational or intraperitoneal routes in either static or ventilated caging. Mice that were exposed to aerosolized B. pseudomallei transmitted the bacterium to approximately 4% of their naïve cagemates, whereas mice that were infected by the intraperitoneal route transmitted to approximately 8% of their naïve cagemates. Whilst the exact route of transmission remains to be determined, the results of this study showed that low levels of mouse-to-mouse transmission of B. pseudomallei are possible. We conclude that although the chance of reinfection is low amongst mice housed in the same cage, this possible scenario should be considered when interpreting data from the BALB/c mouse model of melioidosis in lengthy studies.

Introduction Plague is an ancient disease caused by Yersinia pestis , a widely disseminated Tier 1 pathogen that poses significant public health and biothreat risks. The rapid course and high mortality of pneumonic plague limit the efficacy of antibiotic treatment and mandate the need for an effective, licensed, and readily available vaccine. New candidate vaccines are being developed; however, their efficacy in nonhuman primates, optimal vaccination schedule and immune response, duration of protection, and breadth of coverage against various virulent strains are inadequately understood. In the current work, we explored homologous and heterologous vaccination schemes using the sensitive BALB/c mouse models of bubonic and pneumonic plague challenged with Y. pestis strain C12. This strain, a derivative of the wild-type strain CO92, lacks the anti-phagocytic F1 capsule yet remains highly virulent. Protection against such nonencapsulated strains has been particularly elusive. Methods We tested the efficacy of live attenuated vaccine (LAV) derivatives of Y. pestis CO92 or C12 with a deletion of a type 3 secretion-associated gene (Δ yscN ) or the pgm pigmentation locus, and they were cured of the pPst (PCP1) plasmid (CO92 pgm − pPst − ). The LAVs were evaluated alone or accompanied by a dose of a protein subunit vaccine (rF1V or rV). Results The most protective and immunogenic vaccination scheme, as tested under a variety of conditions in bubonic and pneumonic plague models, was heterologous vaccination with a LAV and the recombinant rF1V or rV protein subunit vaccine. Furthermore, in the heterologous scheme, different LAVs and subunit vaccines could be substituted, affording flexibility in vaccine component selection. We also evaluated a novel intervention strategy consisting of vaccination and post-exposure antibiotic treatment. The layering of vaccination with the LAVs and post-exposure treatment with streptomycin was synergistic, extending the time after the Y. pestis C12 challenge when treatment remained effective and affording a sparing of antibiotics. Conclusion The current work defined effective and flexible vaccination and treatment interventions that successfully prevented lethal infection with virulent, nonencapsulated Y. pestis .

Plague is an ancient disease that continues to be of concern to both the public health and biodefense research communities. Pneumonic plague is caused by hematogenous spread of Yersinia pestis bacteria from a ruptured bubo to the lungs or by directly inhaling aerosolized bacteria. The fatality rate associated with pneumonic plague is significant unless effective antibiotic therapy is initiated soon after an early and accurate diagnosis is made. As with all bacterial pathogens, drug resistance is a primary concern when developing strategies to combat these Yersinia pestis infections in the future. While there has been significant progress in vaccine development, no FDA-approved vaccine strategy exists; thus, other medical countermeasures are needed. Antibody treatment has been shown to be effective in animal models of plague. We produced fully human polyclonal antibodies in transchromosomic bovines vaccinated with the recombinant F1-V plague vaccine. The resulting human antibodies opsonized Y. pestis bacteria in the presence of RAW264.7 cells and afforded significant protection to BALB/c mice after exposure to aerosolized Y. pestis. These data demonstrate the utility of this technology to produce large quantities of non-immunogenic anti-plague human antibodies to prevent or possibly treat pneumonic plague in human.

The microbial pathogens Burkholderia pseudomallei and Bacillus anthracis are unrelated bacteria, yet both are the etiologic agents of naturally occurring diseases in animals and humans and are classified as Tier 1 potential biothreat agents. B. pseudomallei is the gram-negative bacterial agent of melioidosis, a major cause of sepsis and mortality globally in endemic tropical and subtropical regions. B. anthracis is the gram-positive spore-forming bacterium that causes anthrax. Infections acquired by inhalation of these pathogens are challenging to detect early while the prognosis is best; and they possess innate multiple antibiotic resistance or are amenable to engineered resistance. Previous studies showed that the early generation, rarely used aminocoumarin novobiocin was very effective in vitro against a range of highly disparate biothreat agents. The objective of the current research was to begin to characterize the therapeutic efficacy of novobiocin in mouse models of anthrax and melioidosis. The antibiotic was highly efficacious against infections by both pathogens, especially B. pseudomallei. Our results supported the concept that specific older generation antimicrobials can be effective countermeasures against infection by bacterial biothreat agents. Finally, novobiocin was shown to be a potential candidate for inclusion in a combined pre-exposure vaccination and post-exposure treatment strategy designed to target bacterial pathogens refractory to a single medical countermeasure.

Burkholderia pseudomallei and the closely related species, Burkholderia mallei, produce similar multifaceted diseases which range from rapidly fatal to protracted and chronic, and are a major cause of mortality in endemic regions. Besides causing natural infections, both microbes are Tier 1 potential biothreat agents. Antibiotic treatment is prolonged with variable results, hence effective vaccines are urgently needed. The purpose of our studies was to compare candidate vaccines that target both melioidosis and glanders to identify the most efficacious one(s) and define residual requirements for their transition to the non-human primate aerosol model. Studies were conducted in the C57BL/6 mouse model to evaluate the humoral and cell-mediated immune response and protective efficacy of three Burkholderia vaccine candidates against lethal aerosol challenges with B. pseudomallei K96243, B. pseudomallei MSHR5855, and B. mallei FMH. The recombinant vaccines generated significant immune responses to the vaccine antigens, and the live attenuated vaccine generated a greater immune response to OPS and the whole bacterial cells. Regardless of the candidate vaccine evaluated, the protection of mice was associated with a dampened cytokine response within the lungs after exposure to aerosolized bacteria. Despite being delivered by two different platforms and generating distinct immune responses, two experimental vaccines, a capsule conjugate + Hcp1 subunit vaccine and the live B. pseudomallei 668 ΔilvI strain, provided significant protection and were down-selected for further investigation and advanced development.

Burkholderia pseudomallei, the gram-negative bacterium that causes melioidosis, is notoriously difficult to treat with antibiotics. A significant effort has focused on identifying protective vaccine strategies to prevent melioidosis. However, when used as individual medical countermeasures both antibiotic treatments (therapeutics or post-exposure prophylaxes) and experimental vaccine strategies remain partially protective. Here we demonstrate that when used in combination, current vaccine strategies (recombinant protein subunits AhpC and/or Hcp1 plus capsular polysaccharide conjugated to CRM197 or the live attenuated vaccine strain B. pseudomallei 668 ΔilvI) and co-trimoxazole regimens can result in near uniform protection in a mouse model of melioidosis due to apparent synergy associated with distinct medical countermeasures. Our results demonstrated significant improvement when examining several suboptimal antibiotic regimens (e.g., 7-day antibiotic course started early after infection or 21-day antibiotic course with delayed initiation). Importantly, this combinatorial strategy worked similarly when either protein subunit or live attenuated vaccines were evaluated. Layered and integrated medical countermeasures will provide novel treatment options for melioidosis as well as diseases caused by other pathogens that are refractory to individual strategies, particularly in the case of engineered, emerging, or re-emerging bacterial biothreat agents.

The etiologic agent of plague, Yersinia pestis, is a globally distributed pathogen which poses both a natural and adversarial threat. Due largely to the rapid course and high mortality of pneumonic plague, vaccines are greatly needed. Two-component protein vaccines have been unreliable and potentially vulnerable to vaccine resistance. We evaluated the safety and efficacy of eight live Y. pestis strains derived from virulent strains CO92 or KIM6+ and mutated in one or more virulence-associated gene(s) or cured of plasmid pPst. Stringent, single-dose vaccination allowed down-selection of the two safest and most protective vaccine candidates, CO92 mutants pgm- pPst- and ΔyscN. Both completely protected BALB/c mice against subcutaneous and aerosol challenge with Y. pestis. Strain CD-1 outbred mice were more resistant to bubonic (but not pneumonic) plague than BALB/c mice, but the vaccines elicited partial protection of CD-1 mice against aerosol challenge, while providing full protection against subcutaneous challenge. A ΔyscN mutant of the nonencapsulated C12 strain was expected to display antigens previously concealed by the capsule. C12 ΔyscN elicited negligible titers to F1 but comparable antibody levels to whole killed bacteria, as did CO92 ΔyscN. Although one dose of C12 ΔyscN was not protective, vaccination with two doses of either CO92 ΔyscN, or a combination of the ΔyscN mutants of C12 and CO92, protected optimally against lethal bubonic or pneumonic plague. Protection against encapsulated Y. pestis required inclusion of F1 in the vaccine and was associated with high anti-F1 titers.