Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections.

The bacterium Burkholderia pseudomallei is the cause of melioidosis infectious disease. In this bacterium, the BLF1 protein wide inhibits the synthesis of proteins in human cells. This disease is reported to cause a death rate of 40% in some parts of the world. Currently, no effective vaccine is available against this bacterial infection. In this study, therefore, a Nano vaccine was synthesized based on the trimethyl chitosan (TMC) polymer containing the BLF1 recombinant protein, and its immunogenicity and protection in Syrian mice were evaluated by oral and subcutaneous injections. The BLF1 recombinant protein expression was induced in Escherichia coli Bl21 (DE3) and purified by the affinity chromatography technique. Recombinant protein-containing nanoparticles (NPs) were then synthesized by the ionotropic gelation method. After oral and subcutaneous injections, antibody titration was assessed by the indirect ELISA assay. Finally, murine groups were challenged using the BLF1 toxin. The results indicated that the immune system showed more antibody titration in subcutaneous injection than in the oral form. However, the results were reversed in the challenge results, and the survival rate was more significant in the oral injection.

Diabetes and Infectious Diseases with a Focus on Melioidosis.

Diabetes mellitus (DM) leads to impaired innate and adaptive immune responses. This renders individuals with DM highly susceptible to microbial infections such as COVID-19, tuberculosis and melioidosis. Melioidosis is a tropical disease caused by the bacterial pathogen Burkholderia pseudomallei, where diabetes is consistently reported as the most significant risk factor associated with the disease. Type-2 diabetes is observed in 39% of melioidosis patients where the risk of infection is 13-fold higher than non-diabetic individuals. B. pseudomallei is found in the environment and is an opportunistic pathogen in humans, often exhibiting severe clinical manifestations in immunocompromised patients. The pathophysiology of diabetes significantly affects the host immune responses that play a critical role in fighting the infection, such as leukocyte and neutrophil impairment, macrophage and monocyte inhibition and natural killer cell dysfunction. These defects result in delayed recruitment as well as activation of immune cells to target the invading B. pseudomallei. This provides an advantage for the pathogen to survive and adapt within the immunocompromised diabetic patients. Nevertheless, knowledge gaps on diabetes-infectious disease comorbidity, in particular, melioidosis-diabetes comorbidity, need to be filled to fully understand the dysfunctional host immune responses and adaptation of the pathogen under diabetic conditions to guide therapeutic options.

Role of Burkholderia pseudomallei-Specific IgG2 in Adults with Acute Melioidosis, Thailand.

Melioidosis is a life-threatening infectious disease caused by the gram-negative bacillus Burkholderia pseudomallei. An effective vaccine is needed, but data on protective immune responses in human melioidosis are lacking. We used ELISA and an antibody-dependent cellular phagocytosis assay to identify the major features of protective antibodies in patients with acute melioidosis in Thailand. We found that high levels of B. pseudomallei-specific IgG2 are associated with protection against death in a multivariable logistic regression analysis adjusting for age, diabetes, renal disease, and neutrophil count. Serum from melioidosis survivors enhanced bacteria uptake into human monocytes expressing FcγRIIa-H/R131, an intermediate-affinity IgG2-receptor, compared with serum from nonsurvivors. We did not find this enhancement when using monocytes carrying the low IgG2-affinity FcγRIIa-R131 allele. The findings indicate the importance of IgG2 in protection against death in human melioidosis, a crucial finding for antibody-based therapeutics and vaccine development.

Rab32 GTPase, as a direct target of miR-30b/c, controls the intracellular survival of Burkholderia pseudomallei by regulating phagosome maturation.

Burkholderia pseudomallei is a gram-negative, facultative intracellular bacterium, which causes a disease known as melioidosis. Professional phagocytes represent a crucial first line of innate defense against invading pathogens. Uptake of pathogens by these cells involves the formation of a phagosome that matures by fusing with early and late endocytic vesicles, resulting in killing of ingested microbes. Host Rab GTPases are central regulators of vesicular trafficking following pathogen phagocytosis. However, it is unclear how Rab GTPases interact with B. pseudomallei to regulate the transport and maturation of bacterial-containing phagosomes. Here, we showed that the host Rab32 plays an important role in mediating antimicrobial activity by promoting phagosome maturation at an early phase of infection with B. pseudomallei. And we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30c in B. pseudomallei infected macrophages. Subsequently, we showed that B. pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features. And Rab32 enhances phagosome acidification and promotes the fusion of B. pseudomallei-containing phagosomes with lysosomes to activate cathepsin D, resulting in restricted intracellular growth of B. pseudomallei. Additionally, Rab32 mediates phagosome maturation depending on its guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding state. Finally, we report the previously unrecognized role of miR-30b/30c in regulating B. pseudomallei-containing phagosome maturation by targeting Rab32 in macrophages. Altogether, we provide a novel insight into the host immune-regulated cellular pathway against B. pseudomallei infection is partially dependent on Rab32 trafficking pathway, which regulates phagosome maturation and enhances the killing of this bacterium in macrophages.

Gasdermin D Protects from Melioidosis through Pyroptosis and Direct Killing of Bacteria.

Gasdermin D (GSDMD) cleavage by caspase-1 or caspase-11 inflammasomes triggers pyroptosis, a lytic form of cell death protective against intracellular bacteria. In this study, we examine the role of GSDMD in a mouse model of melioidosis. Gsdmd-/- mice were more susceptible than wild-type mice to intranasal infection with Burkholderia thailandensis Production of IL-18, but not IL-1ß, was decreased in Gsdmd-/- infected mice. Despite lower IL-18, IFN-γ was produced in similar amounts in wild-type and Gsdmd-/- mice. In vitro, secretion of both IL-1ß and IL-18 by macrophages or dendritic cells infected with B. thailandensis was dependent on GSDMD. Surprisingly, wild-type or GSDMD-deficient neutrophils secreted similar amounts of IL-1ß, suggesting these cells may be the source of the GSDMD-independent IL-1ß detected in vivo. Recombinant GSDMD was able to directly kill B. thailandensis in vitro upon processing by active caspase-1. Moreover, bacteria harvested from wild-type, but not Gsdmd-/- , macrophages were more susceptible to the microbicidal effect of hydrogen peroxide or human ß-defensin-3. Finally, we provide evidence that pyroptosis of in vitro infected macrophages is directly microbicidal. Taken together, these results indicate that the protective action of GSDMD in melioidosis is primarily due to induction of pyroptosis and direct killing of bacteria rather than production of cytokines.

Caspase-4 Mediates Restriction of Burkholderia pseudomallei in Human Alveolar Epithelial Cells.

Melioidosis is an infectious disease with a high mortality rate responsible for community-acquired sepsis in Southeast Asia and Northern Australia. The causative agent of this disease is Burkholderia pseudomallei, a Gram-negative bacterium that resides in soil and contaminated natural water. After entering into host cells, the bacteria escape into the cytoplasm, which has numerous cytosolic sensors, including the noncanonical inflammatory caspases. Although the noncanonical inflammasome (caspase-11) has been investigated in a murine model of B. pseudomallei infection, its role in humans, particularly in lung epithelial cells, remains unknown. We, therefore, investigated the function of caspase-4 (ortholog of murine caspase-11) in intracellular killing of B. pseudomallei The results showed that B. pseudomallei induced caspase-4 activation at 12 h postinfection in human alveolar epithelial A549 cells. The number of intracellular B. pseudomallei bacteria was increased in the absence of caspase-4, suggesting its function in intracellular bacterial restriction. In contrast, a high level of caspase-4 processing was observed when cells were infected with lipopolysaccharide (LPS) mutant B. pseudomallei The enhanced bacterial clearance in LPS-mutant-infected cells is also correlated with a higher degree of caspase-4 activation. These results highlight the susceptibility of the LPS mutant to caspase-4-mediated intracellular bacterial killing.

Dysregulation of TNF-α and IFN-γ expression is a common host immune response in a chronically infected mouse model of melioidosis when comparing multiple human strains of Burkholderia pseudomallei.

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-1ß, KC, and MIG. By immunohistochemical and in situ RNA hybridization analysis we found that the increased expression of proinflammatory cytokines (IL-1α, IL-1ß, 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.

Diabetes alters immune response patterns to acute melioidosis in humans.

Diabetes mellitus (DM) is a serious global health problem currently affecting over 450 million people worldwide. Defining its interaction with major global infections is an international public health priority. Melioidosis is caused by Burkholderia pseudomallei, an exemplar pathogen for studying intracellular bacterial infection in the context of DM due to the 12-fold increased risk in this group. We characterized immune correlates of survival in peripheral blood of acute melioidosis patients with and without DM and highlight different immune response patterns. We demonstrate the importance of circulating NK cells and show that CX3CR1 expression on lymphocytes is a novel correlate of survival from acute melioidosis. Furthermore, excessive serum levels of IL-15 and IL-18BP contribute to poor outcome independent of DM comorbidity. CD8+ T cells and granzyme B expression in NK cells are important for survival of non-DM patients, whereas high antibody titers against B. pseudomallei and double-negative T cells are linked to survival of DM patients. Recall responses support a role of γδ T-cell-derived IFN-γ in the establishment of protective immunity in the DM group. Defining the hallmarks of protection in people with DM is crucial for the design of new therapies and vaccines targeting this rapidly expanding risk group.

Caspase-11-dependent pyroptosis of lung epithelial cells protects from melioidosis while caspase-1 mediates macrophage pyroptosis and production of IL-18.

Infection with Burkholderia pseudomallei or B. thailandensis triggers activation of the NLRP3 and NLRC4 inflammasomes leading to release of IL-1ß and IL-18 and death of infected macrophages by pyroptosis, respectively. The non-canonical inflammasome composed of caspase-11 is also activated by these bacteria and provides protection through induction of pyroptosis. The recent generation of bona fide caspase-1-deficient mice allowed us to reexamine in a mouse model of pneumonic melioidosis the role of caspase-1 independently of caspase-11 (that was also absent in previously generated Casp1-/- mice). Mice lacking either caspase-1 or caspase-11 were significantly more susceptible than wild type mice to intranasal infection with B. thailandensis. Absence of caspase-1 completely abolished production of IL-1ß and IL-18 as well as pyroptosis of infected macrophages. In contrast, in mice lacking caspase-11 IL-1ß and IL-18 were produced at normal level and macrophages pyroptosis was only marginally affected. Adoptive transfer of bone marrow indicated that caspase-11 exerted its protective action both in myeloid cells and in radio-resistant cell types. B. thailandensis was shown to readily infect mouse lung epithelial cells triggering pyroptosis in a caspase-11-dependent way in vitro and in vivo. Importantly, we show that lung epithelial cells do not express inflammasomes components or caspase-1 suggesting that this cell type relies exclusively on caspase-11 for undergoing cell death in response to bacterial infection. Finally, we show that IL-18's protective action in melioidosis was completely dependent on its ability to induce IFNγ production. In turn, protection conferred by IFNγ against melioidosis was dependent on generation of ROS through the NADPH oxidase but independent of induction of caspase-11. Altogether, our results identify two non-redundant protective roles for caspase-1 and caspase-11 in melioidosis: Caspase-1 primarily controls pyroptosis of infected macrophages and production of IL-18. In contrast, caspase-11 mediates pyroptosis of infected lung epithelial cells.

Utility of endobronchial ultrasound guided trans-bronchial needle aspiration in diagnosis of melioidosis - case series and review of literature.

Endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) is routinely performed for diagnostic evaluation of mediastinal lymphadenopathy due to various etiologies with excellent sensitivity and specificity. Melioidosis can have atypical features like isolated mediastinal lymphadenopathy mimicking as tuberculosis or lymphoma. Differentiation of such atypical melioidosis presentation become difficult due to similar clinical, radiological and even similar EBUS lymph node characteristics. Role of EBUS TBNA in diagnosing melioidosis is under investigated and sparsely reported. We describe two cases of melioidosis diagnosed by point of care rapid lateral flow immunoassay antigen testing and culture of EBUS-TBNA samples from necrotic mediastinal lymph nodes.

Thrombocytopenia Impairs Host Defense Against Burkholderia pseudomallei (Melioidosis).

Background: Infection with the gram-negative bacillus Burkholderia pseudomallei (melioidosis) is an important cause of pneumosepsis in Southeast Asia and has a mortality of up to 40%. We aimed to assess the role of platelets in the host response against B. pseudomallei infection. Methods: Association between platelet counts and mortality was determined in 1160 patients with culture-proven melioidosis. Mice treated with (low- or high-dose) platelet-depleting antibody were inoculated intranasally with B. pseudomallei and killed. Additional studies using functional glycoprotein Ibα-deficient mice were conducted. Results: Thrombocytopenia was present in 31% of patients at admission and predicted mortality in melioidosis patients even after adjustment for confounders. In our murine-melioidosis model, platelet counts decreased, and mice treated with a platelet-depleting antibody showed enhanced mortality and higher bacterial loads compared to mice with normal platelet counts. Low platelet counts had a modest impact on early-pulmonary neutrophil influx. Reminiscent of their role in hemostasis, platelet depletion impaired vascular integrity, resulting in early lung bleeding. Glycoprotein Ibα-deficient mice had reduced platelet counts during B. pseudomallei infection together with an impaired local host defense in the lung. Conclusions: Thrombocytopenia predicts mortality in melioidosis patients and, during experimental melioidosis, platelets play a protective role in both innate immunity and vascular integrity.

Lipopolysaccharides from Different Burkholderia Species with Different Lipid A Structures Induce Toll-Like Receptor 4 Activation and React with Melioidosis Patient Sera.

Lipopolysaccharides (LPSs) of Gram-negative bacteria comprise lipid A, core, and O-polysaccharide (OPS) components. Studies have demonstrated that LPSs isolated from the pathogenic species Burkholderia pseudomallei and Burkholderia mallei and from less-pathogenic species, such as Burkholderia thailandensis, are potent immune stimulators. The LPS structure of B. pseudomallei, the causative agent of melioidosis, is highly conserved in isolates from Thailand; however, the LPSs isolated from other, related species have not been characterized to enable understanding of their immune recognition and antigenicities. Here, we describe the structural and immunological characteristics of the LPSs isolated from eight Burkholderia species and compare those for B. pseudomallei to those for the other seven species. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), gas chromatography (GC), SDS-PAGE, Toll-like receptor 4 (TLR4) stimulation, and immunoblot analysis were performed on these Burkholderia species. MALDI-TOF profiles demonstrated that Burkholderia lipid A contains predominantly penta-acylated species modified with 4-amino-4-deoxy-arabinose residues at both terminal phosphate groups. The lipid A could be differentiated based on mass differences at m/z 1,511, 1,642, 1,773, and 1,926 and on fatty acid composition. LPSs of all species induced TLR4-dependent NF-κB responses; however, while SDS-PAGE analysis showed similar LPS ladder patterns for B. pseudomallei, B. thailandensis, and B. mallei, these patterns differed from those of other Burkholderia species. Interestingly, immunoblot analysis demonstrated that melioidosis patient sera cross-reacted with OPSs of other Burkholderia species. These findings can be used to better understand the characteristics of LPS in Burkholderia species, and they have implications for serological diagnostics based on the detection of antibodies to OPS.

Next-generation Diagnostics for Melioidosis: Evaluation of a Prototype i-STAT Cartridge to Detect Burkholderia pseudomallei Biomarkers.

BACKGROUND: Infection with the gram-negative bacterium Burkholderia pseudomallei can result in melioidosis, a life-threatening disease that can be difficult to diagnose. Culture remains the gold standard for diagnosis but requires laboratory resources not available in many endemic regions. A lateral flow immunoassay has shown promise for POC diagnostics but suffers from low sensitivity when used on blood samples. PCR also has low sensitivity on blood, attributed to the low bacterial numbers in blood observed in melioidosis patients, even when bacteraemic. METHODS: A prototype i-STAT cartridge was developed to utilize the monoclonal antibody specific for the capsule of pathogenic Burkholderia species employed on the LFI. The resulting POC assay was evaluated on 414 clinical specimens from Darwin, Australia and Cambodia. RESULTS: The i-STAT assay accurately distinguished Australian blood culture positive melioidosis patients from Australian patients hospitalized with other infections (AUC = 0.91, 95% CI 0.817 - 1.0). We derived an assay cutoff with 76% sensitivity and 94% specificity that correctly classified 88% (n = 74) of the Australian patients. Interestingly, only 46% (6/13) of the culture-positive melioidosis patients in Cambodia were classified correctly. Of great importance however, the assay detected capsule from blood samples for 32% of blood culture negative melioidosis patients in both cohorts and previously undiagnosed melioidosis patients in Cambodia. In addition the assay showed high sensitivity and specificity for urine, pus and sputum. CONCLUSIONS: Diagnostic tools that are not dependent upon the growth kinetics or the levels of bacteremia of B. pseudomallei represent the next-generation of diagnostics and must be pursued further.

Novel multi-component vaccine approaches for Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis. Historically believed to be a relatively rare human disease in tropical countries, a recent study estimated that, worldwide, there are approximately 165 000 human melioidosis cases per year, more than half of whom die. The bacterium is inherently resistant to many antibiotics and treatment of the disease is often protracted and ineffective. There is no licensed vaccine against melioidosis, but a vaccine is predicted to be of value if used in high-risk populations. There has been progress over the last decade in the pursuit of an effective vaccine against melioidosis. Animal models of disease including mouse and non-human primates have been developed, and these models show that antibody responses play a key role in protection against melioidosis. Surprisingly, although B. pseudomallei is an intracellular pathogen there is limited evidence that CD8+  T cells play a role in protection. It is evident that a multi-component vaccine, incorporating one or more protective antigens, will probably be essential for protection because of the pathogen's sophisticated virulence mechanisms as well as strain heterogeneity. Multi-component vaccines in development include glycoconjugates, multivalent subunit preparations, outer membrane vesicles and other nano/microparticle platforms and live-attenuated or inactivated bacteria. A consistent finding with vaccine candidates tested in mice is the ability to induce sterilizing immunity at low challenge doses and extended time to death at higher challenge doses. Further research to identify ways of eliciting more potent immune responses might provide a path for licensing an effective vaccine.

Melioidosis patient serum-reactive synthetic tetrasaccharides bearing the predominant epitopes of Burkholderia pseudomallei and Burkholderia mallei O-antigens.

Melioidosis and glanders, respectively caused by the Gram-negative bacteria Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), are considered as urgent public health issues in developing countries and potential bioterrorism agents. Bp and Bm lipopolysaccharides (LPS) have been identified as attractive vaccine candidates for the development of prophylactic measures against melioidosis and glanders. Bp and Bm express structurally similar LPSs wherein the O-antigen (OAg) portion consists of a heteropolymer whose repeating unit is a disaccharide composed of d-glucose and 6-deoxy-l-talose residues, the latter being diversely acetylated and methylated. Herein we report the synthesis of two tetrasaccharides mimicking the main substitution epitopes of Bp and Bm LPS OAgs. The assembly of the tetrasaccharides was achieved using a sequential glycosylation strategy while relying on the late-stage epimerization of the inner rhamnose into a 6-deoxy-l-talose residue. We show that these synthetic compounds strongly react with culture-confirmed Thai melioidosis patient serum and closely mimic the antigenicity of native Bp OAg. Our results suggest that these tetrasaccharides could be suitable candidates for the development of vaccines and/or diagnostic tools against melioidosis and glanders.

Polysaccharides from Burkholderia species as targets for vaccine development, immunomodulation and chemical synthesis.

Covering: up to 2018 Burkholderia species are a vast group of human pathogenic, phytopathogenic, and plant- or environment-associated bacteria. B. pseudomallei, B. mallei, and B. cepacia complex are the causative agents of melioidosis, glanders, and cystic fibrosis-related infections, respectively, which are fatal diseases in humans and animals. Due to their high resistance to antibiotics, high mortality rates, and increased infectivity via the respiratory tract, B. pseudomallei and B. mallei have been listed as potential bioterrorism agents by the Centers for Disease Control and Prevention. Burkholderia species are able to produce a large network of surface-exposed polysaccharides, i.e., lipopolysaccharides, capsular polysaccharides, and exopolysaccharides, which are virulence factors, immunomodulators, major biofilm components, and protective antigens, and have crucial implications in the pathogenicity of Burkholderia-associated diseases. This review provides a comprehensive and up-to-date account regarding the structural elucidation and biological activities of surface polysaccharides produced by Burkholderia species. The chemical synthesis of oligosaccharides mimicking Burkholderia polysaccharides is described in detail. Emphasis is placed on the recent research efforts toward the development of glycoconjugate vaccines against melioidosis and glanders based on synthetic or native Burkholderia oligo/polysaccharides.

Antibodies against In Vivo-Expressed Antigens Are Sufficient To Protect against Lethal Aerosol Infection with Burkholderia mallei and Burkholderia pseudomallei.

Burkholderia mallei, a facultative intracellular bacterium and tier 1 biothreat, causes the fatal zoonotic disease glanders. The organism possesses multiple genes encoding autotransporter proteins, which represent important virulence factors and targets for developing countermeasures in pathogenic Gram-negative bacteria. In the present study, we investigated one of these autotransporters, BatA, and demonstrate that it displays lipolytic activity, aids in intracellular survival, is expressed in vivo, elicits production of antibodies during infection, and contributes to pathogenicity in a mouse aerosol challenge model. A mutation in the batA gene of wild-type strain ATCC 23344 was found to be particularly attenuating, as BALB/c mice infected with the equivalent of 80 median lethal doses cleared the organism. This finding prompted us to test the hypothesis that vaccination with the batA mutant strain elicits protective immunity against subsequent infection with wild-type bacteria. We discovered that not only does vaccination provide high levels of protection against lethal aerosol challenge with B. mallei ATCC 23344, it also protects against infection with multiple isolates of the closely related organism and causative agent of melioidosis, Burkholderia pseudomallei Passive-transfer experiments also revealed that the protective immunity afforded by vaccination with the batA mutant strain is predominantly mediated by IgG antibodies binding to antigens expressed exclusively in vivo Collectively, our data demonstrate that BatA is a target for developing medical countermeasures and that vaccination with a mutant lacking expression of the protein provides a platform to gain insights regarding mechanisms of protective immunity against B. mallei and B. pseudomallei, including antigen discovery.

Boosting of post-exposure human T-cell and B-cell recall responses in vivo by Burkholderia pseudomallei-related proteins.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with high incidence and mortality in South East Asia and northern Australia. To date there is no protective vaccine and antibiotic treatment is prolonged and not always effective. Most people living in endemic areas have been exposed to the bacteria and have developed some immunity, which may have helped to prevent disease. Here, we used a humanized mouse model (hu-PBL-SCID), reconstituted with human peripheral blood mononuclear cells from seropositive donors, to illustrate the potential of three known antigens (FliC, OmpA and N-PilO2) for boosting both T-cell and B-cell immune responses. All three antigens boosted the production of specific antibodies in vivo, and increased the number of antibody and interferon-γ-secreting cells, and induced antibody affinity maturation. Moreover, antigen-specific antibodies isolated from either seropositive individuals or boosted mice, were found to enhance phagocytosis and oxidative burst activities from human polymorphonuclear cells. Our study demonstrates that FliC, OmpA and N-PilO2 can stimulate human memory T and B cells and highlight the potential of the hu-PBL-SCID system for screening and evaluation of novel protein antigens for inclusion in future vaccine trials against melioidosis.

BIITE: A Tool to Determine HLA Class II Epitopes from T Cell ELISpot Data.

Activation of CD4+ T cells requires the recognition of peptides that are presented by HLA class II molecules and can be assessed experimentally using the ELISpot assay. However, even given an individual's HLA class II genotype, identifying which class II molecule is responsible for a positive ELISpot response to a given peptide is not trivial. The two main difficulties are the number of HLA class II molecules that can potentially be formed in a single individual (3-14) and the lack of clear peptide binding motifs for class II molecules. Here, we present a Bayesian framework to interpret ELISpot data (BIITE: Bayesian Immunogenicity Inference Tool for ELISpot); specifically BIITE identifies which HLA-II:peptide combination(s) are immunogenic based on cohort ELISpot data. We apply BIITE to two ELISpot datasets and explore the expected performance using simulations. We show this method can reach high accuracies, depending on the cohort size and the success rate of the ELISpot assay within the cohort.