γδ T Cells Mediate Protection against Neutrophil-associated Lung Inflammation in Pulmonary Melioidosis.

Pulmonary melioidosis is a severe tropical infection caused by Burkholderia pseudomallei and is associated with high mortality, despite early antibiotic treatment. γδ T cells have been increasingly implicated as drivers of the host neutrophil response during bacterial pneumonia, but their role in pulmonary melioidosis is unknown. Here, we report that in patients with melioidosis, a lower peripheral blood γδ T-cell concentration is associated with higher mortality, even when adjusting for severity of illness. γδ T cells were also enriched in the lung and protected against mortality in a mouse model of pulmonary melioidosis. γδ T-cell deficiency in infected mice induced an early recruitment of neutrophils to the lung, independent of bacterial burden. Subsequently, γδ T-cell deficiency resulted in increased neutrophil-associated inflammation in the lung as well as impaired bacterial clearance. In addition, γδ T cells influenced neutrophil function and subset diversity in the lung after infection. Our results indicate that γδ T cells serve a novel protective role in the lung during severe bacterial pneumonia by regulating excessive neutrophil-associated inflammation.

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.

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.

Monoclonal Antibodies Opsonize Burkholderia spp. and Reduce Intracellular Actin Tail Formation in a Macrophage Infection Assay.

Melioidosis is a neglected tropical disease caused by the bacterium Burkholderia pseudomallei. The bacterium is intrinsically resistant to various antibiotics, and melioidosis is therefore difficult to treat successfully without a relapse in infection. B. pseudomallei is an intracellular pathogen and therefore, to eradicate the infection, antimicrobials must be able to access bacteria in an intracellular niche. This study assessed the ability of a panel of monoclonal antibodies (MAbs) to opsonize Burkholderia species and determine the effect that each antibody has on bacterial virulence in vitro. Murine macrophage infection assays demonstrated that monoclonal antibodies against the capsule of B. pseudomallei are opsonizing. Furthermore, one of these monoclonal antibodies reduced bacterial actin tail formation in our in vitro assays, indicating that antibodies could reduce the intracellular spread of Burkholderia thailandensis. The data presented in this paper demonstrate that monoclonal antibodies are opsonizing and can decrease bacterial actin tail formation, thus decreasing their intracellular spread. These data have informed selection of an antibody for development of an antibody-antibiotic conjugate (AAC) for melioidosis. IMPORTANCE Melioidosis is difficult to treat successfully due to the causal bacterium being resistant to many classes of antibiotics, therefore limiting available therapeutic options. New and improved therapies are urgently required to treat this disease. Here, we have investigated the potential of monoclonal antibodies to target this intracellular pathogen. We have demonstrated that monoclonal antibodies can target the bacterium, increase uptake into macrophages, and reduce actin tail formation required by the bacterium for spread between cells. Through targeting the bacterium with antibodies, we hope to disarm the pathogen, reducing the spread of infection. Ultimately, we aim to use an opsonizing antibody to deliver antibiotics intracellularly by developing an antibody-antibiotic conjugate therapeutic for melioidosis.

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.

Development of an immunomagnetic separation-ELISA for the detection of Burkholderia pseudomallei in blood samples.

BACKGROUND: Septicemic melioidosis caused by Burkholderia pseudomallei is a serious cause of morbidity and mortality. An effective, rapid and simple diagnostic method is required for detection of B. pseudomallei infection. OBJECTIVE: To develop immunomagnetic beads (IMB) coupled with ELISA (IMB-ELISA) for detection of B. pseudomallei in blood samples of patients with suspected melioidosis. METHODS: For separation of B. pseudomallei from buffer, blood samples and hemoculture, 200 nm immunomagnetic beads (IMBs) coated with 4B11 monoclonal antibody (4B11-IMBs) against exopolysaccharide antigens were used. The detection was done by an ELISA based biotin-streptavidin system. The sensitivity and specificity were evaluated. RESULTS: 4B11-IMBs (100 µg) were successfully developed and used for detection of B. pseudomallei in 1 ml samples. Transmission electron microscopy (TEM) imaging demonstrated B. pseudomallei was captured by 4B11-IMBs. The IMBs showed high capture efficiency (98%) with B. pseudomallei in buffer. The IMB-ELISA assay was highly specific for B. pseudomallei. It showed no cross-reactions with other bacteria, except B. mallei. The limits of the B. pseudomallei assay detection for detecting B. pseudomallei in either buffer solution or blood was 102 CFU/ml. The IMB-ELISA detection sensitivity in blood samples was 44.5%. Although it did not give the highest sensitivity, it was useful for detection with hemoculture that was faster than conventional methods. CONCLUSION: This study suggests the IMB-ELISA assay offers a simple and highly specific method with a turnaround time of 6 h for detection of B. pseudomallei. The developed assay can be applied in hospitals for surveillance of B. pseudomallei.

Burkholderia pseudomallei peri-prosthetic infection following medial malleolar internal fixation: a case report.

BACKGROUND: Melioidosis-associated peri-prosthetic infection is extremely rare. To date, melioidosis associated septic arthritis of the ankle joint following a medial malleolar internal fixation has not been reported. CASE PRESENTATION: We describe a 49-year-old male with a history of long standing diabetes who presented with fever, constitutional symptoms and right ankle pain for 1 week. Ten years ago, he underwent a medial malleolar screw fixation following a traumatic closed fracture. His initial right ankle radiographs showed no evidence of osteomyelitis. He underwent a wound debridement and washout of the right ankle joint. The peripheral blood and pus from the ankle joint was culture positive for Burkholderia pseudomallei with very high antibody titres. His subsequent radiographs showed features of chronic osteomyelitis. He was treated with a prolonged course of antibiotics and repeated wound debridement. At follow up after 6 months, he had no clinical features of recurrent infection. CONCLUSIONS: Melioidosis should be entertained in the differential diagnosis of peri-prosthetic infections in high risk patients.

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.

Anti-Cancer Drug HMBA Acts as an Adjuvant during Intracellular Bacterial Infections by Inducing Type I IFN through STING.

The anti-proliferative agent hexamethylene bisacetamide (HMBA) belongs to a class of hybrid bipolar compounds developed more than 30 y ago for their ability to induce terminal differentiation of transformed cells. Recently, HMBA has also been shown to trigger HIV transcription from latently infected cells, via a CDK9/HMBA inducible protein-1 dependent process. However, the effect of HMBA on the immune response has not been explored. We observed that pretreatment of human peripheral blood mononuclear cells with HMBA led to a markedly increased production of IL-12 and IFN-γ, but not of TNF-α, IL-6, and IL-8 upon subsequent infection with Burkholderia pseudomallei and Salmonella enterica HMBA treatment was also associated with better intracellular bacterial control. HMBA significantly improved IL-12p70 production from CD14+ monocytes during infection partly via the induction of type I IFN in these cells, which primed an increased transcription of the p35 subunit of IL-12p70 during infection. HMBA also increased early type I IFN transcription in human monocytic and epithelial cell lines, but this was surprisingly independent of its previously reported effects on positive transcription elongation factor b and HMBA inducible protein-1. Instead, the effect of HMBA was downstream of a calcium influx, and required the pattern recognition receptor and adaptor STING but not cGAS. Our work therefore links the STING-IRF3 axis to enhanced IL-12 production and intracellular bacterial control in primary monocytes. This raises the possibility that HMBA or related small molecules may be explored as therapeutic adjuvants to improve disease outcomes during intracellular bacterial infections.

Virulence of the Melioidosis Pathogen Burkholderia pseudomallei Requires the Oxidoreductase Membrane Protein DsbB.

The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei A panel of B. pseudomalleidsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.

A Rapid Immunochromatography Test Based on Hcp1 Is a Potential Point-of-Care Test for Serological Diagnosis of Melioidosis.

Melioidosis is a fatal infectious disease caused by the environmental bacterium Burkholderia pseudomallei It is highly endemic in Asia and northern Australia but neglected in many other tropical countries. Melioidosis patients have a wide range of clinical manifestations, and definitive diagnosis requires bacterial culture, which can be time-consuming. A reliable rapid serological tool is greatly needed for disease surveillance and diagnosis. We previously demonstrated by enzyme-linked immunosorbent assay (ELISA) that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we developed a rapid immunochromatography test (ICT) using Hcp1 as the target antigen (Hcp1-ICT). We evaluated this test for specific antibody detection using serum samples obtained from 4 groups of human subjects, including the following: (i) 487 culture-confirmed melioidosis patients from four hospitals in northeast Thailand; (ii) 202 healthy donors from northeast Thailand; (iii) 90 U.S. healthy donors; and (iv) 207 patients infected with other organisms. Compared to culture results as a gold standard, the sensitivity of ICT for all hospitals was 88.3%. The specificities for Thai donors and U.S. donors were 86.1% and 100%, respectively, and the specificity for other infections was 91.8%. The results of the Hcp1-ICT demonstrated 92.4% agreement with the Hcp1-ELISA results with a kappa value of 0.829, indicating that the method is much improved compared with the current serological method, the indirect hemagglutination assay (IHA) (69.5% sensitivity and 67.6% specificity for Thais). The Hcp1-ICT represents a potential point-of-care (POC) test and may be used to replace the IHA for screening of melioidosis in hospitals as well as in resource-limited areas.

Reverse vaccinology and subtractive genomics-based putative vaccine targets identification for Burkholderia pseudomallei Bp1651.

The Burkholderia pseudomallei is a unique bio-threat and causative agent of melioidosis. The B. pseudomallei Bp1651 strain has been isolated from a chronic cystic fibrosis patient. The genome-level DNA sequences information of this strain has recently been published. Unfortunately, there is no commercial vaccine available till date to combat B. pseudomallei infection. The genome-wide prioritization approaches are widely used for the identification of potential therapeutic candidates against pathogens. In the present study, we utilized the recently available annotated genomic information of B. pseudomallei Bp1651 through subtractive genomics and reverse-vaccinology strategies to identify its potential vaccine targets. The analyses identified more than 60 pathogen-specific, human host non-homologous proteins that may prioritize in future studies to investigate therapeutic targets for B. pseudomallei Bp1651. The potential B and T-cells antigenic determinant peptides from these pathogen-specific proteins were cataloged using antigenicity and epitope prediction tools. The analyses unveiled a promising antigenic peptide "FQWEFSLSV" from protein-export membrane protein (SecF) of Bp1651 strain, which was predicted to interact with multiple class I and class II MHC alleles with IC50 value < 100 nM. The molecular docking analysis verified favorable molecular interaction of this lead antigenic peptide with the ligand-binding pocket residues of HLA A*02:06 human host immune cell surface receptor. This peptide is predicted to be a suitable epitope capable to elicit the cell-mediated immune response against the B. pseudomallei pathogen. The putative epitopes and proteins identified in this study may be promising vaccine targets against Bp1651 as well as other pathogenic strains of B. pseudomallei.

Differential Production of Type I IFN Determines the Reciprocal Levels of IL-10 and Proinflammatory Cytokines Produced by C57BL/6 and BALB/c Macrophages.

Pattern recognition receptors detect microbial products and induce cytokines, which shape the immunological response. IL-12, TNF-α, and IL-1ß are proinflammatory cytokines, which are essential for resistance against infection, but when produced at high levels they may contribute to immunopathology. In contrast, IL-10 is an immunosuppressive cytokine, which dampens proinflammatory responses, but it can also lead to defective pathogen clearance. The regulation of these cytokines is therefore central to the generation of an effective but balanced immune response. In this study, we show that macrophages derived from C57BL/6 mice produce low levels of IL-12, TNF-α, and IL-1ß, but high levels of IL-10, in response to TLR4 and TLR2 ligands LPS and Pam3CSK4, as well as Burkholderia pseudomallei, a Gram-negative bacterium that activates TLR2/4. In contrast, macrophages derived from BALB/c mice show a reciprocal pattern of cytokine production. Differential production of IL-10 in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages was due to a type I IFN and ERK1/2-dependent, but IL-27-independent, mechanism. Enhanced type I IFN expression in LPS-stimulated C57BL/6 macrophages was accompanied by increased STAT1 and IFN regulatory factor 3 activation. Furthermore, type I IFN contributed to differential IL-1ß and IL-12 production in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages via both IL-10-dependent and -independent mechanisms. These findings highlight key pathways responsible for the regulation of pro- and anti-inflammatory cytokines in macrophages and reveal how they may differ according to the genetic background of the host.

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.