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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 bimA Bm 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, 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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: Yersinia 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., Methods: In 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 - Δ caf1 , live Y. pestis pgm- pPst - Δ caf1 /Δ yopD , or recombinant F1-V (rF1-V) combined with adjuvants., Results: The 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., Conclusions: This 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Davies, Biryukov, Rill, Klimko, Hunter, Dankmeyer, Miller, Shoe, Mlynek, Talyansky, Toothman, Qiu, Bozue and Cote.)

Francisella tularensis is one of the several biothreat agents for which a licensed vaccine is needed. To ensure vaccine protection is achieved across a range of virulent F. tularensis strains, we assembled and characterized a panel of F. tularensis isolates to be utilized as challenge strains. A promising tularemia vaccine candidate is rLVS Δ capB / iglABC (rLVS), in which the vector is the LVS strain with a deletion in the capB gene and which additionally expresses a fusion protein comprising immunodominant epitopes of proteins IglA, IglB, and IglC. Fischer rats were immunized subcutaneously 1-3 times at 3-week intervals with rLVS at various doses. The rats were exposed to a high dose of aerosolized Type A strain Schu S4 (FRAN244), a Type B strain (FRAN255), or a tick derived Type A strain (FRAN254) and monitored for survival. All rLVS vaccination regimens including a single dose of 10 7 CFU rLVS provided 100% protection against both Type A strains. Against the Type B strain, two doses of 10 7 CFU rLVS provided 100% protection, and a single dose of 10 7 CFU provided 87.5% protection. In contrast, all unvaccinated rats succumbed to aerosol challenge with all of the F. tularensis strains. A robust Th1-biased antibody response was induced in all vaccinated rats against all F. tularensis strains. These results demonstrate that rLVS Δ capB / iglABC provides potent protection against inhalational challenge with either Type A or Type B F. tularensis strains and should be considered for further analysis as a future tularemia vaccine.

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.

One pathogen that commonly causes gastrointestinal illnesses from the consumption of contaminated food is Escherichia coli O157:H7. In 2011 in Germany, however, there was a prominent outbreak of bloody diarrhea with a high incidence of hemolytic uremic syndrome (HUS) caused by an atypical, more virulent E. coli O104:H4 strain. To facilitate the identification of this lesser-known, atypical E. coli O104:H4 strain, we wanted to identify phenotypic differences between it and a strain of O157:H7 in different media and culture conditions. We found that E. coli O104:H4 strains produced considerably more biofilm than the strain of O157:H7 at 37 °C ( p = 0.0470-0.0182) Biofilm production was significantly enhanced by the presence of 5% CO 2 ( p = 0.0348-0.0320). In our study on the innate immune response to the E. coli strains, we used HEK293 cells that express Toll-like receptors (TLRs) 2 or 4. We found that E. coli O104:H4 strains had the ability to grow in a novel HEK293 cell culture medium, while the E. coli O157:H7 strain could not. Thus, we uncovered previously unknown phenotypic properties of E. coli O104:H4 to further differentiate this pathogen from E. coli O157:H7.

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.

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 Δ ilvI strain, provided significant protection and were down-selected for further investigation and advanced development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Biryukov, Cote, Klimko, Dankmeyer, Rill, Shoe, Hunter, Shamsuddin, Velez, Hedrick, Rosario-Acevedo, Talyansky, Schmidt, Orne, Fetterer, Burtnick, Brett, Welkos and DeShazer.)

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 Δ ilvI ) 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Klimko, Shoe, Rill, Hunter, Dankmeyer, Talyansky, Schmidt, Orne, Fetterer, Biryukov, Burtnick, Brett, DeShazer and Cote.)

Successful bacterial pathogens have evolved to avoid activating an innate immune system in the host that responds to the pathogen through distinct Toll-like receptors (TLRs). The general class of biochemical components that activate TLRs has been studied extensively, but less is known about how TLRs interact with the class of compounds that are still associated with the live pathogen. Accordingly, we examined the activation of surface assembled TLR 2, 4, and 5 with live Tier 1 Gram-negative pathogens that included Yersinia pestis (plague), Burkholderia mallei (glanders), Burkholderia pseudomallei (melioidosis), and Francisella tularensis (tularemia). We found that Y. pestis CO92 grown at 28°C activated TLR2 and TLR4, but at 37°C the pathogen activated primarily TLR2. Although B. mallei and B. pseudomallei are genetically related, the former microorganism activated predominately TLR4, while the latter activated predominately TLR2. The capsule of wild-type B. pseudomallei 1026b was found to mitigate the activation of TLR2 and TLR4 when compared to a capsule mutant. Live F. tularensis (Ft) Schu S4 did not activate TLR2 or 4, although the less virulent Ft LVS and F. novicida activated only TLR2. B. pseudomallei purified flagellin or flagella attached to the microorganism activated TLR5. Activation of TLR5 was abolished by an antibody to TLR5, or a mutation of fli C, or elimination of the pathogen by filtration. In conclusion, we have uncovered new properties of the Gram-negative pathogens, and their interaction with TLRs of the host. Further studies are needed to include other microorganism to extend our observations with their interaction with TLRs, and to the possibility of leading to new efforts in therapeutics against these pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amemiya, Dankmeyer, Bernhards, Fetterer, Waag, Worsham and DeShazer.)

Burkholderia pseudomallei is an infectious bacterium of clinical and biodefense concern, and is the causative agent of melioidosis. The mortality rate can reach up to 50% and affects 165,000 people per year; however, there is currently no vaccine available. In this study, we examine the antigen-specific immune response to a vaccine formulated with antigens derived from an outer membrane protein in B. pseudomallei , Bucl8. Here, we employed a number of bioinformatic tools to predict Bucl8-derived epitopes that are non-allergenic and non-toxic, but would elicit an immune response. From these data, we formulated a vaccine based on two extracellular components of Bucl8, the β-barrel loops and extended collagen and non-collagen domains. Outbred CD-1 mice were immunized with vaccine formulations-composed of recombinant proteins or conjugated synthetic peptides with adjuvant-to assess the antigen-specific immune responses in mouse sera and lymphoid organs. We found that mice vaccinated with either Bucl8-derived components generated a robust TH2-skewed antibody response when antigen was combined with the adjuvant AddaVax, while the TH1 response was limited. Mice immunized with synthetic loop peptides had a stronger, more consistent antibody response than recombinant protein antigens, based on higher IgG titers and recognition of bacteria. We then compared peptide-based vaccines in an established C57BL/6 inbred mouse model and observed a similar TH2-skewed response. The resulting formulations will be applied in future studies examining the protection of Bucl8-derived vaccines.

Relatively recent advances in plague vaccinology have produced the recombinant fusion protein F1-V plague vaccine. This vaccine has been shown to readily protect mice from both bubonic and pneumonic plague. The protection afforded by this vaccine is solely based upon the immune response elicited by the F1 or V epitopes expressed on the F1-V fusion protein. Accordingly, questions remain surrounding its efficacy against infection with non-encapsulated (F1-negative) strains. In an attempt to further optimize the F1-V elicited immune response and address efficacy concerns, we examined the inclusion of multiple toll-like receptor agonists into vaccine regimens. We examined the resulting immune responses and also any protection afforded to mice that were exposed to aerosolized Yersinia pestis . Our data demonstrate that it is possible to further augment the F1-V vaccine strategy in order to optimize and augment vaccine efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Biryukov, Dankmeyer, Shamsuddin, Velez, Rill, Rosario-Acevedo, Klimko, Shoe, Hunter, Ward, Cazares, Fetterer, Bozue, Worsham, Cote and Amemiya.)

Burkholderia mallei is a gram-negative obligate animal pathogen that causes glanders, a highly contagious and potentially fatal disease of solipeds including horses, mules, and donkeys. Humans are also susceptible, and exposure can result in a wide range of clinical forms, i.e., subclinical infection, chronic forms with remission and exacerbation, or acute and potentially lethal septicemia and/or pneumonia. Due to intrinsic antibiotic resistance and the ability of the organisms to survive intracellularly, current treatment regimens are protracted and complicated; and no vaccine is available. As a consequence of these issues, and since B. mallei is infectious by the aerosol route, B. mallei is regarded as a major potential biothreat agent. To develop optimal medical countermeasures and diagnostic tests, well characterized animal models of human glanders are needed. The goal of this study was to perform a head-to-head comparison of models employing three commonly used nonhuman primate (NHP) species, the African green monkey (AGM), Rhesus macaque, and the Cynomolgus macaque. The natural history of infection and in vitro clinical, histopathological, immunochemical, and bacteriological parameters were examined. The AGMs were the most susceptible NHP to B. mallei; five of six expired within 14 days. Although none of the Rhesus or Cynomolgus macaques succumbed, the Rhesus monkeys exhibited abnormal signs and clinical findings associated with B. mallei infection; and the latter may be useful for modeling chronic B. mallei infection. Based on the disease progression observations, gross and histochemical pathology, and humoral and cellular immune response findings, the AGM appears to be the optimal model of acute, lethal glanders infection. AGM models of infection by B. pseudomallei, the etiologic agent of melioidosis, have been characterized recently. Thus, the selection of the AGM species provides the research community with a single NHP model for investigations on acute, severe, inhalational melioidosis and glanders., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 Δ yscN . 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 Δ yscN mutant of the nonencapsulated C12 strain was expected to display antigens previously concealed by the capsule. C12 Δ yscN elicited negligible titers to F1 but comparable antibody levels to whole killed bacteria, as did CO92 Δ yscN . Although one dose of C12 Δ yscN was not protective, vaccination with two doses of either CO92 Δ yscN, or a combination of the Δysc N 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.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a major cause of sepsis and mortality in endemic regions of Southeast Asia and Northern Australia. B. pseudomallei is a potential bioterrorism agent due to its high infectivity, especially via inhalation, and its inherent resistance to antimicrobials. There is currently no vaccine for melioidosis and antibiotic treatment can fail due to innate drug resistance, delayed diagnosis and treatment, or insufficient duration of treatment. A well-characterized animal model that mimics human melioidosis is needed for the development of new medical countermeasures. This study first characterized the disease progression of melioidosis in the African green monkey (AGM) and rhesus macaque (RM) for non-human primate model down-selection. All AGMs developed acute lethal disease similar to that described in human acute infection following exposure to aerosolized B. pseudomallei strain HBPUB10134a. Only 20% of RMs succumbed to acute disease. Disease progression, immune response and pathology of two other strains of B. pseudomallei, K96243 and MSHR5855, were also compared using AGMs. These three B. pseudomallei strains represent a highly virulent strain from Thailand (HBPUB101034a), a highly virulent strains from Australia (MSHR5855), and a commonly used laboratory strains originating from Thailand (K96243). Animals were observed for clinical signs of infection and blood samples were analyzed for cytokine responses, blood chemistry and leukocyte changes in order to characterize bacterial infection. AGMs experienced fever after exposure to aerosolized B. pseudomallei at the onset of acute disease. Inflammation, abscesses and/or pyogranulomas were observed in lung with all three strains of B. pseudomallei. Inflammation, abscesses and/or pyogranulomas were observed in lymph nodes, spleen, liver and/or kidney with B. pseudomallei, HBPUB10134a and K96243. Additionally, the Australian strain MSHR5855 induced brain lesions in one AGM similar to clinical cases of melioidosis seen in Australia. Elevated serum levels of IL-1β, IL-1 receptor antagonist, IL-6, MCP-1, G-CSF, HGF, IFNγ, MIG, I-TAC, and MIP-1β at terminal end points can be significantly correlated with non-survivors with B. pseudomallei infection in AGM. The AGM model represents an acute model of B. pseudomallei infection for all three strains from two geographical locations and will be useful for efficacy testing of vaccines and therapeutics against melioidosis. In summary, a dysregulated immune response leading to excessive persistent inflammation and inflammatory cell death is the key driver of acute melioidosis. Early intervention in these pathways will be necessary to counter B. pseudomallei and mitigate the pathological consequences of melioidosis., Competing Interests: The authors have declared that no competing interests exist.

Plague is a zoonotic disease that is caused by Yersinia pestis . Monoclonal antibodies (mAbs) that bind to the V-antigen, a virulence factor that is produced by Y. pestis , can passively protect mice from plague. An analysis of protective mAbs that bind to V-antigen was made to assess binding sites, avidities, and affinities. Anti-V mAbs were screened for their efficacy in a murine model of plague. Antigen-binding sites of protective V mAbs were determined with a linear peptide library, V-antigen fragment, competitive binding, and surface plasmon resonance. The avidities to the V-antigen was determined by ELISA, and affinities of the mAbs to the V-antigen were determined by surface plasmon resonance. The most protective mAb 7.3 bound to a unique conformational site on the V-antigen, while a less protective mAb bound to a different conformational site located on the same V-antigen fragment as mAb 7.3. The avidity of mAb 7.3 for the V-antigen was neither the strongest overall nor did it have the highest affinity for the V-antigen. The binding site of the most protective mAb was critical in its ability to protect against a lethal plague challenge.

Burkholderia pseudomallei and B. mallei are Gram-negative, facultative intracellular bacteria that cause melioidosis and glanders, respectively. Currently, there are no vaccines for these two diseases. Animal models have been developed to evaluate vaccines and therapeutics. Tissues from infected animals, however, must be fixed in formalin and embedded in paraffin (FFPE) before analysis. A brownish staining material in infected tissues that represents the exopolysaccharide of the pathogen was seen by bright field microscopy but not the actual microorganism. Because of these results, FFPE tissue was examined by laser scanning confocal microscopy (LSCM) in an attempt to see the microorganism. Archival FFPE tissues were examined from ten mice, and five nonhuman primates after exposure to B. pseudomallei or B. mallei by LSCM. Additionally, a historical spleen biopsy from a human suspected of exposure to B. mallei was examined. B. pseudomallei was seen in many of the infected tissues from mice. Four out of five nonhuman primates were positive for the pathogen. In the human sample, B. mallei was seen in pyogranulomas in the spleen biopsy. Thus, the presence of the pathogen was validated by LSCM in murine, nonhuman primate, and human FFPE tissues.

Background: Melioidosis is endemic in Southeast Asia and Northern Australia and is caused by the Gram-negative, facultative intracellular pathogen Burkholderia pseudomallei. Diagnosis of melioidosis is often difficult because of the protean clinical presentation of the disease, and it may mimic other diseases, such as tuberculosis. There are many different strains of B. pseudomallei that have been isolated from patients with melioidosis, but it was not clear if they could cause a similar disease in a chronic BALB/c murine model of melioidosis. Hence, we wanted to examine chronically infected mice exposed to different strains of B. pseudomallei to determine if there were differences in the host immune response to the pathogen., Results: We identified common host immune responses exhibited in chronically infected BALB/c mice, although there was some heterogeneity in the host response in chronically infected mice after exposure to different strains of B. pseudomallei. They all displayed pyogranulomatous lesions in their spleens with a large influx of monocytes/macrophages, NK cells, and neutrophils identified by flow cytometry. Sera from chronically infected mice by ELISA exhibited elevated IgG titers to the pathogen, and we detected by Luminex micro-bead array technology a significant increase in the expression of inflammatory cytokines/chemokines, such as IFN-γ, IL-1α, IL-1β, KC, and MIG. By immunohistochemical and in situ RNA hybridization analysis we found that the increased expression of proinflammatory cytokines (IL-1α, IL-1β, TNF-α, IFN-γ) was confined primarily to the area with the pathogen within pyogranulomatous lesions. We also found that cultured splenocytes from chronically infected mice could express IFN-γ, TNF-α, and MIP-1α ex vivo without the need for additional exogenous stimulation. In addition by flow cytometry, we detected significant amounts of intracellular expression of TNF-α and IFN-γ without a protein transport blocker in monocytes/macrophages, NK cells, and neutrophils but not in CD4 + or CD8 + T cells in splenocytes from chronically infected mice., Conclusion: Taken together the common features we have identified in chronically infected mice when 10 different human clinical strains of B. pseudomallei were examined could serve as biomarkers when evaluating potential therapeutic agents in mice for the treatment of chronic melioidosis in humans.

The administration of antipyretic analgesics prior to, in conjunction with, or due to sequelae associated with vaccination is a common yet somewhat controversial practice. In the context of human vaccination, it is unclear if even short-term analgesic regimens can significantly alter the resulting immune response, as literature exists to support several scenarios including substantial immune interference. In this report, we used a live attenuated Yersinia pestis vaccine to examine the impact of analgesic administration on the immune response elicited by a single dose of a live bacterial vaccine in mice. Mice were assessed by evaluating natural and provoked behavior, as well as food and water consumption. The resulting immune responses were assessed by determining antibody titers against multiple antigens and assaying cellular responses in stimulated splenocytes collected from vaccinated animals. We observed no substantial benefit to the mice associated with the analgesic administration. Splenocytes from both C57BL/6 and BALB/c vaccinated mice receiving acetaminophen have a significantly reduced interferon-gamma (IFN-γ) recall response. Additionally, there is a significantly lower immunoglobulin (Ig)G2a/IgG1 ratio in vaccinated BALB/c mice treated with either acetaminophen or meloxicam and a significantly lower IgG2c/IgG1 ratio in vaccinated C57BL/6 mice treated with acetaminophen. Taken together, our data indicate that the use of analgesics, while possibly ethically warranted, may hinder the accurate characterization and evaluation of novel vaccine strategies with little to no appreciable benefits to the vaccinated mice., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Melioidosis is an emerging disease that is caused by the facultative intracellular pathogen Burkholderia pseudomallei . It is intrinsically resistant to many antibiotics and host risk factors play a major role in susceptibility to infection. Currently, there is no human or animal vaccine against melioidosis. In this study, multiple B. pseudomallei MSHR668 deletion mutants were evaluated as live attenuated vaccines in the sensitive BALB/c mouse model of melioidosis. The most efficacious vaccines after an intraperitoneal challenge with 50-fold over the 50% median lethal dose (MLD 50 ) with B. pseudomallei K96243 were 668 Δ hisF and 668 Δ ilvI . Both vaccines completely protected mice in the acute phase of infection and showed significant protection (50% survivors) during the chronic phase of infection. The spleens of the survivors that were examined were sterile. Splenocytes from mice vaccinated with 668 Δ hisF and 668 Δ ilvI expressed higher amounts of IFN-γ after stimulation with B. pseudomallei antigens than splenocytes from mice vaccinated with less protective candidates. Finally, we demonstrate that 668 Δ hisF is nonlethal in immunocompromised NOD/SCID mice. Our results show that 668 Δ hisF and 668 Δ ilvI provide protective cell-mediated immune responses in the acute phase of infection and promote long term survival in the sensitive BALB/c mouse model of melioidosis.

Introduction: Burkholderia mallei (B. mallei) and Burkholderia pseudomallei (B. pseudomallei), causative agents of glanders and melioidosis, respectively, are invasive intracellular pathogens that actively multiply in phagocytic and non-phagocytic cells. Activation of cell-autonomous autophagy mechanism eliminate intracellular pathogens in which p62 a cytosolic cargo protein is selectively degraded, and an accumulation of this marker occurs if autophagy is deficient. Recurrent, relapsed and reinfection of B. pseudomallei in melioidosis patients in endemic area indicative of lack of complete of clearance and persistence of the pathogen. Reasoning that abundance in the levels of p62 may provide an indication of the intracellular infection, we sought to examine whether increase in intracellular p62 and bacterial burden with Burkholderia infection are linked to autophagy deficiency., Methods: In this study, we investigated cell culture and mouse models of disease to identify an association between autophagy biomarkers (p62/NBR1) accumulation and intracellular persistence of B. mallei and B. pseudomallei., Results: We demonstrate, that elevated levels of intracellular p62/NBR1 correlated with bacterial persistence, while pre-treatment with a pharmacological inducer of autophagy, rapamycin, reduced both intracellular p62, and bacterial survival. Our results showed an elevated p62 levels (2-5 fold) in spleen and liver cells of Burkholderia-infected BALB/c mice, as well as in spleen cells of Burkholderia-infected C57BL/6 mice, suggesting that an increase in p62/NBR1 was due to an autophagy deficiency. Similar to p62, cytosolic LC3-I levels were also elevated, while the characteristic conversion to the autophagosome-associated membrane bound form LC3-II was low in spleens of the infected mice further supporting the conclusion that autophagy was deficient., Conclusion: Taken together, our results suggest that an increase in intracellular p62/NBR1 may be a potential host cell biomarker of B. mallei or B. pseudomallei infections, and identifying autophagy manipulation may potentially aid to therapeutic approach for complete clearance of the pathogen., (© 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

Mouse models have been essential to generate supporting data for the research of infectious diseases. Burkholderia pseudomallei, the etiological agent of melioidosis, has been studied using mouse models to investigate pathogenesis and efficacy of novel medical countermeasures to include both vaccines and therapeutics. Previous characterization of mouse models of melioidosis have demonstrated that BALB/c mice present with an acute infection, whereas C57BL/6 mice have shown a tendency to be more resistant to infection and may model chronic disease. In this study, either BALB/c or C57BL/6 mice were exposed to aerosolized human clinical isolates of B. pseudomallei. The bacterial strains included HBPUB10134a (virulent isolate from Thailand), MSHR5855 (virulent isolate from Australia), and 1106a (relatively attenuated isolate from Thailand). The LD50 values were calculated and serial sample collections were performed in order to examine the bacterial burdens in tissues, histopathological features of disease, and the immune response mounted by the mice after exposure to aerosolized B. pseudomallei. These data will be important when utilizing these models for testing novel medical countermeasures. Additionally, by comparing highly virulent strains with attenuated isolates, we hope to better understand the complex disease pathogenesis associated with this bacterium., Competing Interests: The authors have declared that no competing interests exist.

Melioidosis is a neglected tropical disease that is caused by the bacterium Burkholderia pseudomallei and is underreported in many countries where the disease is endemic. A long and costly administration of antibiotics is needed to clear infections, and there is an unmet need for biomarkers to guide antibiotic treatment and increase the number of patients that complete therapy. We identified calprotectin as a lead biomarker of B. pseudomallei infections and examined correlations between this serum protein and the antibiotic treatment outcomes of patients with melioidosis. Serum levels of calprotectin and C-reactive protein were significantly higher in patients with melioidosis and nonmelioidosis sepsis than in healthy controls. Median calprotectin levels were higher in patients with melioidosis than in those with nonmelioidosis sepsis, whereas C-reactive protein levels were similar in both groups. Notably, intensive intravenous antibiotic treatment of patients with melioidosis resulted in lower levels of calprotectin and C-reactive protein ( P < 0.0001), coinciding with recovery. The median percent reduction of calprotectin and C-reactive protein was 71% for both biomarkers after antibacterial therapy. In contrast, we found no significant differences in calreticulin levels between the two melioidosis treatment phases. Thus, reductions in serum calprotectin levels were linked to therapeutic responses to antibiotics. Our results suggest that calprotectin may be a sensitive indicator of melioidosis disease activity and illustrate the potential utility of this biomarker in guiding the duration of antibiotic therapy., (Copyright © 2017 American Society for Microbiology.)

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a Gram negative bacterium designated as a Tier 1 threat. This bacterium is known to be endemic in Southeast Asia and Northern Australia and can infect humans and animals by several routes. Inhalational melioidosis has been associated with monsoonal rains in endemic areas and is also a significant concern in the biodefense community. There are currently no effective vaccines for B. pseudomallei and antibiotic treatment can be hampered by non-specific symptomology and also the high rate of naturally occurring antibiotic resistant strains. Well-characterized animal models will be essential when selecting novel medical countermeasures for evaluation prior to human clinical trials. Here, we further characterize differences between the responses of BALB/c and C57BL/6 mice when challenged with low doses of a low-passage and well-defined stock of B. pseudomallei K96243 via either intraperitoneal or aerosol routes of exposure. Before challenge, mice were implanted with a transponder to collect body temperature readings, and daily body weights were also recorded. Mice were euthanized on select days for pathological analyses and determination of the bacterial burden in selected tissues (blood, lungs, liver, and spleen). Additionally, spleen homogenate and sera samples were analyzed to better characterize the host immune response after infection with aerosolized bacteria. These clinical, pathological, and immunological data highlighted and confirmed important similarities and differences between these murine models and exposure routes.

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

Burkholderia pseudomallei is the etiologic agent of melioidosis, which is endemic in Southeast Asia and Northern Australia. We previously found by the intraperitoneal (IP) route that we could discern differences in virulence in mice amongst different strains of B. pseudomallei. We report an early immune response study comparing two strains in our collection which represent the least, B. pseudomallei 1106a, and one of the most, HBPUB10134a, virulent strains in BALB/c mice. B. pseudomallei HBPUB10134a infected mouse spleens contained a 2-3 log higher bacterial burden than mice infected with B. pseudomallei 1106a 3 days post-infection (PI). More and higher amounts of inflammatory cytokines/chemokines were detected in sera and spleen extracts from B. pseudomallei HBPUB10134a than B. pseudomallei 1106a infected mice. The most prominent were IFNγ, IL-1α, IL-1β, IL-6, IL-10, IP-10, and MIG. After 7 days PI, there was a decrease in bacterial burden in spleens from 1106a infected mice and a decrease in cytokines/chemokines in sera and spleen extracts from both sets of mice. By day 14 PI we saw an increase in monocytes/macrophages, NK cells, and granulocytes in spleens from both sets of mice. No B. pseudomallei HBPUB10134a infected mice survived after this time. In summary, B. pseudomallei HBPUB10134a was more virulent and induced host innate immune responses typical of a more acute-type infection than did B. pseudomallei 1106a which produced a more chronic infection in mice., (Published by Elsevier Ltd.)

The current candidate vaccine against Yersinia pestis infection consists of two subunit proteins: the capsule protein or F1 protein and the low calcium response V protein or V-antigen. Little is known of the recognition of the vaccine by the host's innate immune system and how it affects the acquired immune response to the vaccine. Thus, we vaccinated Toll-like receptor (Tlr) 2, 4, and 2/4-double deficient, as well as signal adaptor protein Myd88-deficient mice. We found that Tlr4 and Myd88 appeared to be required for an optimal immune response to the F1-V vaccine but not Tlr2 when compared to wild-type mice. However, there was a difference between the requirement for Tlr4 and MyD88 in vaccinated animals. When F1-V vaccinated Tlr4 mutant (lipopolysaccharide tolerant) and Myd88-deficient mice were challenged by aerosol with Y. pestis CO92, all but one Tlr4 mutant mice survived the challenge, but no vaccinated Myd88-deficient mice survived the challenge. Spleens from these latter nonsurviving mice showed that Y. pestis was not cleared from the infected mice. Our results suggest that MyD88 appears to be important for both an optimal immune response to F1-V and in protection against a lethal challenge of Y. pestis CO92 in F1-V vaccinated mice.

In the future, we may be faced with the need to provide treatment for an emergent biological threat against which existing vaccines and drugs have limited efficacy or availability. To prepare for this eventuality, our objective was to use a metabolic network-based approach to rapidly identify potential drug targets and prospectively screen and validate novel small-molecule antimicrobials. Our target organism was the fully virulent Francisella tularensis subspecies tularensis Schu S4 strain, a highly infectious intracellular pathogen that is the causative agent of tularemia and is classified as a category A biological agent by the Centers for Disease Control and Prevention. We proceeded with a staggered computational and experimental workflow that used a strain-specific metabolic network model, homology modeling and X-ray crystallography of protein targets, and ligand- and structure-based drug design. Selected compounds were subsequently filtered based on physiological-based pharmacokinetic modeling, and we selected a final set of 40 compounds for experimental validation of antimicrobial activity. We began screening these compounds in whole bacterial cell-based assays in biosafety level 3 facilities in the 20th week of the study and completed the screens within 12 weeks. Six compounds showed significant growth inhibition of F. tularensis, and we determined their respective minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished bacterial growth at 13 µM, with putative activity against pantetheine-phosphate adenylyltransferase, an enzyme involved in the biosynthesis of coenzyme A, encoded by gene coaD. The novel antimicrobial compounds identified in this study serve as starting points for lead optimization, animal testing, and drug development against tularemia. Our integrated in silico/in vitro approach had an overall 15% success rate in terms of active versus tested compounds over an elapsed time period of 32 weeks, from pathogen strain identification to selection and validation of novel antimicrobial compounds.

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.