CD4+ T-cell immunity to the Burkholderia pseudomallei ABC transporter LolC in melioidosis

Burkholderia pseudomallei causes melioidosis, a disease with a wide range of possible outcomes, from seroconversion and dormancy to sepsis and death. This spectrum of host-pathogen interactions poses challenging questions about the heterogeneity in immunity to B. pseudomallei. Models show protection to be dependent on CD4(+) cells and IFN-γ, but little is known about specific target antigens. Having previously implicated the ABC transporter, LolC, in protective immunity, we here use epitope prediction, HLA-binding studies, HLA-transgenic models and studies of T cells from seropositive individuals to characterize HLA-restricted LolC responses. Immunized mice showed long-lasting memory to the protein, whereas predictive algorithms identified epitopes within LolC that subsequently demonstrated strong HLA class II binding. Immunization of HLA-DR transgenics with LolC stimulated T-cell responses to four of these epitopes. Furthermore, the responsiveness of HLA transgenics to LolC revealed a hierarchy supportive of HLA polymorphism-determined differential susceptibility. Seropositive human donors of diverse HLA class II types showed T-cell responses to LolC epitopes, which are conserved among Burkholderia species including Burkholderia cenocepacia, associated with life-threatening cepacia complex in cystic fibrosis patients and Burkholderia mallei, which causes glanders. These findings suggest a role for LolC epitopes in multiepitope vaccine design for melioidosis and related diseases.     

Immunoblot analysis to demonstrate antigenic variability of clinical isolated. Pseudomonas pseudomallei.

Pseudomonas pseudomallei (Ps.ps.) is the causative organism of melioidosis, and is widely distributed in Southeast Asia and Northern Australia. Clinical manifestations range from subclinical infection to fulminant septicemia. To demonstrate the antigenic variability of Ps.ps., 62 clinical isolates from 31 blood, 13 sputum, 9 pus, 3 urine and 6 body fluid culture specimens were studied by SDS-PAGE and immunoblotting. In SDS-PAGE, there were approximately 20 antigenic components with molecular weights ranging from 14 to 66 kilodaltons (KD) which suggested that there was antigenic variability among these 62 clinical isolates of Ps.ps. Attempts to correlate immunoblot profiles with clinical illness or sources of specimens were not successful but 6 common antigens were identified with molecular weight of 17.5, 21, 33, 34, 40 and 45 KD, respectively. Among these antigens, the 45 KD component was recognised by all patients' sera. Thus, the 45 KD protein antigen may be useful for the future approach in immunodiagnosis of melioidosis.     

A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens

Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.     

Characterization of monoclonal antibodies to protein antigen of Salmonella typhi

Two monoclonal antibodies were produced against protein antigens of Salmonella typhi. One of the antibodies (STP14) belongs to the immunoglobulin G1K subclass, and the other (STP13) was assigned to the immunoglobulin G2a(kappa) subclass. Both antibodies could recognize the 34.0-kilodalton protein antigen from S. typhi. The specificity of these antibodies was tested by immunoblotting with a panel of crude protein antigens from 12 bacteria causing enteric fever and enteric fever-like illness: S. typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, S. choleraesuis, S. enteritidis, S. krefeld, S. panama, S. typhimurium, Escherichia coli, Pseudomonas pseudomallei, and Yersinia enterocolitica. In a modified double-antibody sandwich enzyme-linked immunosorbent assay they could detect the protein antigen at ca. 0.6 microgram/ml. These monoclonal antibodies should be of great value in the diagnostic test for detecting S. typhi antigen in samples of bodily fluids isolated from patients with typhoid fever and in studies of the chemical structure and other immunological properties of this 34.0-kilodalton protein.     

Human polymorphonuclear neutrophil responses to Burkholderia pseudomallei in healthy and diabetic subjects

The major predisposing factor for melioidosis is diabetes mellitus, but no immunological mechanisms have been investigated to explain this. In this study, polymorphonuclear neutrophil (PMN) responses to Burkholderia pseudomallei, the causative agent of melioidosis, in healthy and diabetic Thai subjects were determined by flow cytometry. The results showed that B. pseudomallei displayed reduced uptake by PMNs compared to Salmonella enterica serovar Typhimurium and Escherichia coli. Additionally, intracellular survival of B. pseudomallei was detected throughout a 24-h period, indicating the intrinsic resistance of B. pseudomallei to killing by PMNs. Moreover, PMNs from diabetic subjects displayed impaired phagocytosis of B. pseudomallei, reduced migration in response to interleukin-8, and an inability to delay apoptosis. These data show that B. pseudomallei is intrinsically resistant to phagocytosis and killing by PMNs. These observations, together with the impaired migration and apoptosis in diabetes mellitus, may explain host susceptibility in melioidosis.     

Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

Burkholderia pseudomallei is the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing of B. pseudomallei remains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response to B. pseudomallei in a dose- and time-dependent manner. B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways. B. pseudomallei mutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.     

Role of T cells in innate and adaptive immunity against murine Burkholderia pseudomallei infection

Antigen-specific T cells are important sources of interferon (IFN)-gamma for acquired immunity to intracellular pathogens, but they can also produce IFN- gamma directly via a "bystander" activation pathway in response to proinflammatory cytokines. We investigated the in vivo role of cytokine- versus antigen-mediated T cell activation in resistance to the pathogenic bacterium Burkholderia pseudomallei. IFN-gamma, interleukin (IL)-12, and IL-18 were essential for initial bacterial control in infected mice. B. pseudomallei infection rapidly generated a potent IFN-gamma response from natural killer (NK) cells, NK T cells, conventional T cells, and other cell types within 16 h after infection, in an IL-12- and IL-18-dependent manner. However, early T cell- and NK cell-derived IFN-gamma responses were functionally redundant in cell depletion studies, with IFN-gamma produced by other cell types, such as major histocompatibility complex class II(int) F4/80(+) macrophages being sufficient for initial resistance. In contrast, B. pseudomallei-specific CD4(+) T cells played an important role during the later stage of infection. Thus, the T cell response to primary B. pseudomallei infection is biphasic, an early cytokine-induced phase in which T cells appear to be functionally redundant for initial bacterial clearance, followed by a later antigen-induced phase in which B. pseudomallei-specific T cells, in particular CD4(+) T cells, are important for host resistance.