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.

BpOmpW Antigen Stimulates the Necessary Protective T-Cell Responses Against Melioidosis.

Melioidosis is a potentially fatal bacterial disease caused by Burkholderia pseudomallei and is estimated to cause 89,000 deaths per year in endemic areas of Southeast Asia and Northern Australia. People with diabetes mellitus are most at risk of melioidosis, with a 12-fold increased susceptibility for severe disease. Interferon gamma (IFN-γ) responses from CD4 and CD8 T cells, but also from natural killer (NK) and natural killer T (NKT) cells, are necessary to eliminate the pathogen. We previously reported that immunization with B. pseudomallei OmpW (BpOmpW antigen) protected mice from lethal B. pseudomallei challenge for up to 81 days. Elucidating the immune correlates of protection of the protective BpOmpW vaccine is an essential step prior to clinical trials. Thus, we immunized either non-insulin-resistant C57BL/6J mice or an insulin-resistant C57BL/6J mouse model of type 2 diabetes (T2D) with a single dose of BpOmpW. BpOmpW induced strong antibody responses, stimulated effector CD4+ and CD8+ T cells and CD4+ CD25+ Foxp3+ regulatory T cells, and produced higher IFN-γ responses in CD4+, CD8+, NK, and NKT cells in non-insulin-resistant mice. The T-cell responses of insulin-resistant mice to BpOmpW were comparable to those of non-insulin-resistant mice. In addition, as a precursor to its evaluation in human studies, humanized HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice elicited IFN-γ recall responses in an enzyme-linked immune absorbent spot (ELISpot)-based study. Moreover, human donor peripheral blood mononuclear cells (PBMCs) exposed to BpOmpW for 7 days showed T-cell proliferation. Finally, plasma from melioidosis survivors with diabetes recognized our BpOmpW vaccine antigen. Overall, the range of approaches used strongly indicated that BpOmpW elicits the necessary immune responses to combat melioidosis and bring this vaccine closer to clinical trials.

Evaluation of antigen-detecting and antibody-detecting diagnostic test combinations for diagnosing melioidosis.

BACKGROUND: Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in many tropical developing countries and has a high mortality. Here we evaluated combinations of a lateral flow immunoassay (LFI) detecting B. pseudomallei capsular polysaccharide (CPS) and enzyme-linked immunosorbent assays (ELISA) detecting antibodies against hemolysin co-regulated protein (Hcp1) or O-polysaccharide (OPS) for diagnosing melioidosis. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon-sepsis). Cases included 192 patients with a clinical specimen culture positive for B. pseudomallei. Controls included 502 patients who were blood culture positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae or were polymerase chain reaction assay positive for malaria or dengue. Serum samples collected within 24 hours of admission were stored and tested using a CPS-LFI, Hcp1-ELISA and OPS-ELISA. When assessing diagnostic tests in combination, results were considered positive if either test was positive. We selected ELISA cut-offs corresponding to a specificity of 95%. Using a positive cut-off OD of 2.912 for Hcp1-ELISA, the combination of the CPS-LFI and Hcp1-ELISA had a sensitivity of 67.7% (130/192 case patients) and a specificity of 95.0% (477/502 control patients). The sensitivity of the combination (67.7%) was higher than that of the CPS-LFI alone (31.3%, p<0.001) and that of Hcp1-ELISA alone (53.6%, p<0.001). A similar phenomenon was also observed for the combination of CPS-LFI and OPS-ELISA. In case patients, positivity of the CPS-LFI was associated with a short duration of symptoms, high modified Sequential (sepsis-related) Organ Failure Assessment (SOFA) score, bacteraemia and mortality outcome, while positivity of Hcp1-ELISA was associated with a longer duration of symptoms, low modified SOFA score, non-bacteraemia and survival outcome. CONCLUSIONS/SIGNIFICANCE: A combination of antigen-antibody diagnostic tests increased the sensitivity of melioidosis diagnosis over individual tests while preserving high specificity. Point-of-care tests for melioidosis based on the use of combination assays should be further developed and evaluated.

Melioidosis Patient Survival Correlates With Strong IFN-γ Secreting T Cell Responses Against Hcp1 and TssM.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a serious infectious disease with diverse clinical manifestations. The morbidity and mortality of melioidosis is high in Southeast Asia and no licensed vaccines currently exist. This study was aimed at evaluating human cellular and humoral immune responses in Thai adults against four melioidosis vaccine candidate antigens. Blood samples from 91 melioidosis patients and 100 healthy donors from northeast Thailand were examined for immune responses against B. pseudomallei Hcp1, AhpC, TssM and LolC using a variety of cellular and humoral immune assays including IFN-γ ELISpot assays, flow cytometry and ELISA. PHA and a CPI peptide pool were also used as control stimuli in the ELISpot assays. Hcp1 and TssM stimulated strong IFN-γ secreting T cell responses in acute melioidosis patients which correlated with survival. High IFN-γ secreting CD4+ T cell responses were observed during acute melioidosis. Interestingly, while T cell responses of melioidosis patients against the CPI peptide pool were low at the time of enrollment, the levels increased to the same as in healthy donors by day 28. Although high IgG levels against Hcp1 and AhpC were detected in acute melioidosis patients, no significant differences between survivors and non-survivors were observed. Collectively, these studies help to further our understanding of immunity against disease following natural exposure of humans to B. pseudomallei as well as provide important insights for the selection of candidate antigens for use in the development of safe and effective melioidosis subunit vaccines.

Hijacking of the Host's Immune Surveillance Radars by Burkholderia pseudomallei.

Burkholderia pseudomallei (B. pseudomallei) causes melioidosis, a potentially fatal disease for which no licensed vaccine is available thus far. The host-pathogen interactions in B. pseudomallei infection largely remain the tip of the iceberg. The pathological manifestations are protean ranging from acute to chronic involving one or more visceral organs leading to septic shock, especially in individuals with underlying conditions similar to COVID-19. Pathogenesis is attributed to the intracellular ability of the bacterium to 'step into' the host cell's cytoplasm from the endocytotic vacuole, where it appears to polymerize actin filaments to spread across cells in the closer vicinity. B. pseudomallei effectively evades the host's surveillance armory to remain latent for prolonged duration also causing relapses despite antimicrobial therapy. Therefore, eradication of intracellular B. pseudomallei is highly dependent on robust cellular immune responses. However, it remains ambiguous why certain individuals in endemic areas experience asymptomatic seroconversion, whereas others succumb to sepsis-associated sequelae. Here, we propose key insights on how the host's surveillance radars get commandeered by B. pseudomallei.

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.

Multicomponent Gold-Linked Glycoconjugate Vaccine Elicits Antigen-Specific Humoral and Mixed TH1-TH17 Immunity, Correlated with Increased Protection against Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal disease with a high mortality rate. The intrinsic resistance to commonly used antibiotics combined with the complex bacterial life cycle has hampered the development of preventive and therapeutic interventions and vaccines. Furthermore, the need of humoral and cell-mediated immunity in protection against B. pseudomallei has complicated the development of effective vaccines. Antigen delivery vaccine platforms that promote humoral and cellular responses while maintaining a safe profile are a roadblock to developing subunit vaccines against intracellular pathogens. Gold nanoparticles (AuNPs) were used for the delivery of multicomponent antigens with the goal of inducing vaccine-mediated immunity, promoting protection against melioidosis disease. Different nanoglycoconjugates using predicted immunogenic protein candidates, Hcp1, FlgL, OpcP, OpcP1, OmpW, and hemagglutinin, were covalently coupled to AuNPs, together with the lipopolysaccharide (LPS) from Burkholderia thailandensis, which acted as an additional antigen. Animals immunized with individually coupled (AuNP-protein-LPS) formulations containing OpcP or OpcP1, together with CpG as an adjuvant, showed a significant increase in protection, whereas a nanovaccine combination (AuNP-Combo2-LPS) showed significant and complete protection against a lethal intranasal B. pseudomallei challenge. Animals immunized with AuNP-Combo2-LPS showed robust humoral antigen-specific (IgG and IgA) responses with higher IgG2c titer, indicating a TH1-skewed response and promotion of macrophage uptake. In addition, immunization with the nanovaccine combination resulted in a mixed antigen-specific TH1-TH17 cytokine profile after immunization. This study provides the basis for an elegant and refined multicomponent glycoconjugate vaccine formulation capable of eliciting both humoral and cell-mediated responses against lethal B. pseudomallei challenge. IMPORTANCE Melioidosis is a complex human disease associated with a wide range of complications caused by the Gram-negative bacillus Burkholderia pseudomallei. The global burden of melioidosis is estimated to have 165,000 cases per year and 89,000 fatal outcomes. The endemicity of B. pseudomallei includes a wide range of tropical regions in Asia, Africa, Latin America, and Australia. Therefore, a viable alternative to prevent human infections is the development of an effective vaccine; however, no approved vaccine for human use is available. This study provides a vaccine strategy against B. pseudomallei and an immune-stimulatory platform to induce strong humoral and T-cell-mediated immunity.

Identification of defective early immune responses to Burkholderia pseudomallei infection in a diet-induced murine model of type 2 diabetes.

Co-occurrence of bacterial infections with type 2 diabetes (T2D) is a global problem. Melioidosis caused by Burkholderia pseudomallei is 10 times more likely to occur in patients with T2D, than in normoglycemic individuals. Using an experimental model of T2D, we observed that greater susceptibility in T2D was due to differences in proportions of infiltrating leucocytes and reduced levels of MCP-1, IFN-γ and IL-12 at sites of infection within 24 h post-infection. However, by 72 h the levels of inflammatory cytokines and bacteria were markedly higher in visceral tissue and blood in T2D mice. In T2D, dysregulated early immune responses are responsible for the greater predisposition to B. pseudomallei infection.

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 sepsis with osteoarticular melioidosis of the hip in a patient with diabetes mellitus.

Melioidosis is caused by the tropical soil pathogen Burkholderia pseudomallei Infection, usually in the form of pneumonia, disproportionately affects people with a risk factor for immune dysregulation and mortality remains high even with treatment. Climate change and increasing rates of diabetes render the populations of endemic areas increasingly vulnerable to the disease, which is emerging as a serious global health threat. We present here a case of a 68-year-old man from northern Australia with sepsis and osteoarticular melioidosis of the hip, and explore the links between diabetes mellitus and melioidosis, particularly with respect to musculoskeletal infection.

Burkholderia pseudomallei triggers canonical inflammasome activation in a human primary macrophage-based infection model.

Most of the current knowledge on Burkholderia pseudomallei-induced inflammasome activation and cell death in macrophages is derived from murine systems. Little is known about the involved bacterial structures and mechanisms in primary human macrophages. This is of particular relevance since murine and human macrophages as well as primary cells and cell lines differ in many aspects of inflammasome activation, including the proteins involved in the recognition of bacterial patterns. In this study, we therefore aimed (i) to establish an in vitro B. pseudomallei infection model with human monocyte-derived primary macrophages from single donors as these cells more closely resemble macrophages in the human host and (ii) to analyze B. pseudomallei-triggered cell death and bacterial elimination in those cells. Our results show that B. pseudomallei-infected primary human macrophages not only release the inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-α, but are also engaged in canonical inflammasome activation as evidenced by caspase-1 and gasdermin D processing. Absence of the B. pseudomallei T3SS-3 needle protein BsaL, a potent activator of the canonical inflammasome, abolished lytic cell death, reduced IL-1ß release, and caspase-1 and gasdermin D processing. IFN-γ, known to promote non-canonical inflammasome activation, did not influence pyroptosis induction or IL-1ß release from infected primary human macrophages. Nevertheless, it reduced intracellular B. pseudomallei loads, an effect which was partially antagonist by the inhibition of NADPH oxidase. Overall, our data implicate T3SS-3 dependent inflammasome activation and IFN-γ induced immune mechanisms as critical defense mechanisms of human macrophages against B. pseudomallei. In addition, our infection model provides a versatile tool to study human host-pathogen interactions and has the potential to elucidate the role of human individual genetic variations in B. pseudomallei infections.

Prediction of Burkholderia pseudomallei DsbA substrates identifies potential virulence factors and vaccine targets.

Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two approaches to predict virulence factors of Burkholderia pseudomallei, the Gram-negative bacterium that causes melioidosis. B. pseudomallei is naturally antibiotic resistant and there are no clinically available melioidosis vaccines. To identify B. pseudomallei protein targets for drug discovery and vaccine development, we chose to search for substrates of the B. pseudomallei periplasmic disulfide bond forming protein A (DsbA). DsbA introduces disulfide bonds into extra-cytoplasmic proteins and is essential for virulence in many Gram-negative organism, including B. pseudomallei. The first approach to identify B. pseudomallei DsbA virulence factor substrates was a large-scale genomic analysis of 511 unique B. pseudomallei disease-associated strains. This yielded 4,496 core gene products, of which we hypothesise 263 are DsbA substrates. Manual curation and database screening of the 263 mature proteins yielded 81 associated with disease pathogenesis or virulence. These were screened for structural homologues to predict potential B-cell epitopes. In the second approach, we searched the B. pseudomallei genome for homologues of the more than 90 known DsbA substrates in other bacteria. Using this approach, we identified 15 putative B. pseudomallei DsbA virulence factor substrates, with two of these previously identified in the genomic approach, bringing the total number of putative DsbA virulence factor substrates to 94. The two putative B. pseudomallei virulence factors identified by both methods are homologues of PenI family ß-lactamase and a molecular chaperone. These two proteins could serve as high priority targets for future B. pseudomallei virulence factor characterization.

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.

Combating the great mimicker: latest progress in the development of Burkholderia pseudomallei vaccines.

Introduction Burkholderia pseudomallei is an environmental intracellular Gram-negative bacterium that causes melioidosis, a severe infectious disease affecting humans and animals. An increase in melioidosis cases worldwide and the high mortality rate of the disease makes it a public health concern. Melioidosis is known as the 'great mimicker' because it presents with a wide range of disease manifestations. B. pseudomallei is naturally resistant to antibiotics and delay in diagnosis leads to ineffective treatment. Furthermore, there is no approved vaccine to prevent melioidosis infection in humans. Therefore, it is a priority to license a vaccine that can be used for both high-risk endemic areas and for biodefense purposes. Areas covered In this review, we have focussed on recent progress in the USA for the development and advancement of lead B. pseudomallei vaccine candidate(s) ready for testing in pre-clinical trials. Those candidates include live-attenuated vaccines, glycoconjugate vaccines, outer-membrane vesicles, and gold nanoparticle vaccines. Expert opinion Side-by-side comparison of the leading B. pseudomallei vaccine candidates will provide important information to further advance studies into pre-clinical trials. The likelihood of any of these current vaccines becoming the selected candidate that will reduce the occurrence of melioidosis worldwide is closer than ever.

Immune responses in beta-thalassaemia: heme oxygenase 1 reduces cytokine production and bactericidal activity of human leucocytes.

Patients with beta-thalassaemia increase the risk of bacterial infections, particularly Burkholderia pseudomallei (Bp), the causative agent of melioidosis in Thailand. Impaired immune cell functions may be the cause of this susceptibility, but detailed mechanisms have not been defined. In this study, we observed impaired production of IFN-gamma and IL-10 by whole blood from beta-thalassaemia patients upon stimulation with a range of bacteria-derived stimuli. In contrast, IFN-gamma response via TCR and plasma IgG specific for Bp were still intact. Importantly, mRNA expression of heme oxygenase 1 (HO-1), a potential modulator of immune function, was increased in whole blood from beta-thalassaemia patients, either with or without stimulation with Bp in vitro. Induction of HO-1 by hemin or CoPP in vitro reduced production of IFN-gamma and IL-10 from healthy human PBMCs and decreased bacterial clearance activity of whole blood from healthy controls and beta-thalassaemia, while inhibition of HO-1 by SnPP enhanced both functions in healthy controls. These results were confirmed to some extent in purified human monocytes of healthy controls. Our results suggest a mechanism that excess hemin of beta-thalassaemia patients is a significant cause of immune suppression via HO-1 induction and may underlie the susceptibility of these individuals to severe bacterial infection.

In vitro passage alters virulence, immune activation and proteomic profiles of Burkholderia pseudomallei.

Serial passage is a problem among many bacterial species, especially those where strains have been stored (banked) for several decades. Prior to banking with an organization such as ATCC, many bacterial strains were passaged for many years, so the characteristics of each strain may be extremely different. This is in addition to any differences in the original host environment. For Burkholderia pseudomallei, the number of serial passages should be carefully defined for each experiment because it undergoes adaptation during the course of serial passages. In the present study, we found that passaged B. pseudomallei fresh clinical isolates and reference strain in Luria-Bertani broth exhibited increased plaque formation, invasion, intracellular replication, Galleria mellonella killing abilities, and cytokine production of host cells. These bacteria also modulated proteomic profiles during in vitro passage. We presume that the modulation of protein expression during in vitro passage caused changes in virulence and immunogenicity phenotypes. Therefore, we emphasize the need for caution regarding the use of data from passaged B. pseudomallei. These findings of phenotypic adaptation during in vitro serial passage can help researchers working on B. pseudomallei and on other species to better understand disparate findings among strains that have been reported for many years.

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.

Development of an immunoassay using recombinant outer membrane protein A and flagellin for diagnosis of goats with melioidosis.

Among domestic animals, melioidosis is one of the most common diseases reported in goat, sheep, and swine. To evaluate the specific antibodies in goats with melioidosis, we developed a serology test using recombinant outer membrane protein A (OmpA) and flagellin (FliC) of Burkholderia pseudomallei as antigens. DNA corresponding to each antigen was cloned into a pET32a vector and expressed in Escherichia coli. Essentially, the recombinant OmpA and FliC were expressed in a soluble form that could be isolated with 95% homogeneity. Both recombinants could be recognized by rabbit antibodies prepared against heat-inactivated B. pseudomallei (1:1,000) on a Western blot. Subsequently, we demonstrated that both recombinants could capture the antibodies present in goat with naturally occurring melioidosis (optimized titer 1:40) while not cross-reacting with the serum samples of goats naturally infected by Corynebacterium pseudotuberculosis or Staphylococcus aureus. Finally, an enzyme-linked immunosorbent assay (ELISA) using 20 goat serum samples without melioidosis and 10 goat serum samples with melioidosis demonstrated that the infected group has significantly higher antibody titer levels than the normal group (P<0.001) when using either OmpA or FliC as an antigen. However, the sensitivity (100%) of the assay using OmpA was superior to that (90%) from using FliC. Serological tests that are commonly used often rely on antigens from crude cell extracts, which pose risks for laboratory-acquired infections and inconsistency in their preparation; however, use of recombinant OmpA is safe; it can potentially be used as a reagent in testing for goat melioidosis.

A DNA Vaccine in Which the RSV-F Ectodomain Is Covalently Linked to the Burkholderia pseudomallei Antigens TssM and Hcp1 Augments the Humoral and Cytotoxic Response in Mice.

DNA vaccines have great potential to control infectious disease, particularly those caused by intracellular organisms. They are inexpensive to produce and can be quickly modified to combat emerging infectious threats, but often fail to generate a strong immunologic response limiting enthusiasm for their use in humans and animals. To improve the immunogenic response, we developed a DNA vaccine in which the F protein ectodomain of Respiratory Syncytial Virus (RSV-F) was covalently linked to specific antigens of interest. The presence of the RSV-F ectodomain allowed secretion of the translated fusion product out of the originally transfected cells followed by its active binding to adjacent cells. This allowed the targeting of a greater number of cells than those originally transfected, enhancing both humoral and cytotoxic immune responses against the expressed antigen(s). We developed an engrafted mouse model that used antigen-expressing tumor cells to assess the in vivo cytotoxic immune response to specific antigens. We then used this model to demonstrate that a DNA vaccine in which the RSV-F ectodomain is fused to two antigens expressed by Burkholderia pseudomallei, the intracellular gram-negative organism that causes melioidosis, generated a stronger cytotoxic response than a DNA vaccine that lacked the RSV-F sequence while still generating a robust humoral response.