Susceptibility to Burkholderia pseudomallei is associated with host immune responses involving tumor necrosis factor receptor-1 (TNFR1) and TNF receptor-2 (TNFR2).

Melioidosis is caused by the facultative intracellular bacterium, Burkholderia pseudomallei. Using C57BL/6 mice, we investigated the role of macrophages, TNF-alpha, TNF receptor-1 (TNFR1) and TNF receptor-2 (TNFR2) in host defense against B. pseudomallei using an experimental model of melioidosis. This study has demonstrated that in vivo depletion of macrophages renders C57BL/6 mice highly susceptible to intranasal infection with B. pseudomallei, with significant mortality occurring within 5 days of infection. Using knockout mice, we have also shown that TNF-alpha and both TNFR1 and TNFR2 are required for optimal control of B. pseudomallei infection. Compared with control mice, increased bacterial loads were demonstrated in spleen and liver of knockout mice at day 2 postinfection, correlating with increased inflammatory infiltrates comprised predominantly of neutrophils and widespread necrosis. Following infection with B. pseudomallei, mortality rates of 85.7%, 70% and 91.7% were observed for mice deficient in TNF-alpha, TNFR1 and TNFR2, respectively. Comparison of survival, bacterial loads and histology indicate that macrophages, TNF-alpha, TNFR1 or TNFR2 play a role in controlling rapid dissemination of B. pseudomallei.

Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents.

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.

A model of immunity to Burkholderia pseudomallei: unique responses following immunization and acute lethal infection.

Burkholderia pseudomallei, the etiological agent of melioidosis, causes significant mortality in endemic regions, but little is known regarding the immune mechanisms required for successful protective immunity. To establish a model of immunization that could be used to study this we screened a library of B. pseudomallei strains for immunogenicity in mice. BALB/c mice were immunized with test strains, and 2 weeks later were given a lethal challenge (LC) of virulent B. pseudomallei. Among 49 strains tested, a single strain, CL04, exhibited strong immunoprotective capacity. Interestingly, CL04 had been cultured from a patient with chronic colonization of B. pseudomallei, which is a rare phenomenon. Mice immunized with 0.1 x LD50 (5 x 10(3) CFU) of CL04 had significantly better survival and lower bacterial loads after LC compared to naïve controls. Dose-response analysis demonstrated more robust immunity after higher immunizing doses, and bacterial inactivation by gamma irradiation diminished the protective effect, indicating a requirement for viable organism for immunity. CL04-induced immunity was demonstrated both in B. pseudomallei-susceptible BALB/c and -resistant C57BL/6 mice. We investigated the gene profile of CL04-induced immunity by analyzing responses to immunization using cDNA microarray. Unique responses involving granulocyte macrophage colony stimulating factor (GM-CSF), the proapoptotic regulator Bad and cyclin-dependent kinase (CDK5) were detected in immunized mice, but these responses were absent in naïve-LC mice. Further, responses differed between mouse strains, indicating dependence on host genetic background. This model will be useful in identifying elements of the immune response required for successful adaptive immunity against B. pseudomallei.

Seropositivity to Burkholderia pseudomallei does not reflect the development of cell-mediated immunity.

Cell-mediated immunity to Burkholderia pseudomallei, the causative agent of melioidosis, provides protection from disease progression. An indirect haemagglutination assay was used to detect antibodies to B. pseudomallei in 1500 healthy donors in an endemic region of Australia. Lymphocyte proliferation, activation and cytokine expression to B. pseudomallei antigen were determined in eight donors who were seropositive and in eight age- and sex-matched controls. In North Queensland, 2.5% of the population was seropositive for B. pseudomallei, which is less than half that which was previously described. Of clinical significance was the observation that while 75% of the seropositive individuals had increased lymphocyte proliferation to B. pseudomallei antigens, there were no significant differences observed in lymphocyte activation or production of cytokines.

Development of protective immunity in a murine model of melioidosis is influenced by the source of Burkholderia pseudomallei antigens.

Melioidosis is a potentially fatal disease caused by the bacterium, Burkholderia pseudomallei. The current study was carried out to determine the mechanisms involved in the development of protective immunity in a murine model of melioidosis. Following intravenous infection with B. pseudomallei, both C57BL/6 and BALB/c mice demonstrated delayed-type hypersensitivity responses and lymphocyte proliferation towards B. pseudomallei antigens, indicating the generation of B. pseudomallei-specific lymphocytes. Adoptive transfer of these lymphocytes to naïve C57BL/6 mice was demonstrated by a delayed-type hypersensitivity response. Mice were not protected from a subsequent lethal challenge with a highly virulent strain of B. pseudomallei, suggesting that a single intravenous dose of the bacterium is insufficient to induce a protective adaptive immune response. Attempts to induce resistance in susceptible BALB/c mice used repetitive low-dose exposure to live B. pseudomallei. Immune responses and resistance following subcutaneous immunization with live B. pseudomallei were compared with exposure to heat-killed, culture filtrate and sonicated B. pseudomallei antigens. Compared to heat-killed B. pseudomallei, significant protection was generated in BALB/c mice following immunization with live bacteria. Our studies also demonstrate that the type of immune response generated in vivo is influenced by the antigenic preparation of B. pseudomallei used for immunization.

Analogous cytokine responses to Burkholderia pseudomallei strains contrasting in virulence correlate with partial cross-protection in immunized mice.

Cytokine mRNA levels were assessed in Burkholderia pseudomallei-susceptible BALB/c mice and B. pseudomallei-resistant C57BL/6 mice following administration of a sublethal dose of less virulent (LV) B. pseudomallei, a candidate immunogen tested for protection against a highly virulent (HV) challenge. Compared on the basis of the bacterial loads, the cytokine patterns induced by HV and LV B. pseudomallei were similar, involving gamma interferon, interleukin-10, and other cytokines. Partial cross-protection between B. pseudomallei strains is shown to be associated with cytokine profiles involving both type 1 and type 2 cytokines.

Demonstration of a cell-mediated immune response in melioidosis.

Melioidosis is a bacterial infection caused by Burkholderia pseudomallei. The aim of this study was to determine whether a cell-mediated adaptive immune response against B. pseudomallei developed in patients who had recovered from melioidosis. Lymphocyte proliferation assays were done on peripheral blood mononuclear cells from patients (n=13) and control subjects (n=10) to determine the lymphocyte response to B. pseudomallei antigens. Production of interferon-gamma and interleukin-10 was also determined. Activation of T cell subsets was assessed by fluorescence-activated cell sorter analysis, using antibodies to CD4, CD8, and CD69 antigens. Lymphocyte proliferation and interferon-gamma production in response to B. pseudomallei antigens were significantly higher (P<.001 for both) in patients than in control subjects. There was also an increase in the percentage of activated CD4+ (P<.004) and activated CD8+ T cells (P<.035) in cell cultures from patients. The development of such a cell-mediated immune response in patients may be essential for their survival.