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.     

Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens

The bacterium Burkholderia pseudomallei causes a life-threatening disease called melioidosis. In vivo experiments in mice have identified that a rapid IFN-gamma response is essential for host survival. To identify the cellular sources of IFN-gamma, spleen cells from uninfected mice were stimulated with B. pseudomallei in vitro and assayed by ELISA and flow cytometry. Costaining for intracellular IFN-gamma vs cell surface markers demonstrated that NK cells and, more surprisingly, CD8(+) T cells were the dominant sources of IFN-gamma. IFN-gamma(+) NK cells were detectable after 5 h and IFN-gamma(+) CD8(+) T cells within 15 h after addition of bacteria. IFN-gamma production by both cell populations was inhibited by coincubation with neutralizing mAb to IL-12 or IL-18, while a mAb to TNF had much less effect. Three-color flow cytometry showed that IFN-gamma-producing CD8(+) T cells were of the CD44(high) phenotype. The preferential activation of NK cells and CD8(+) T cells, rather than CD4(+) T cells, was also observed in response to Listeria monocytogenes or a combination of IL-12 and IL-18 both in vitro and in vivo. This rapid mechanism of CD8(+) T cell activation may be an important component of innate immunity to intracellular pathogens.     

Effect of acidic pH on the invasion efficiency and the type III secretion system of <Emphasis Type="Italic">Burkholderia thailandensis</Emphasis>

Burkholderia thailandensis is a close relative of Burkholderia pseudomallei. These organisms are very similar, but B. thailandensis is far less virulent than B. pseudomallei. Nucleotide sequencing and analysis of 14 B. thailandensis isolates revealed variation in the regions coding for the type III secreted BipD protein. The degree of B. thailandensis BipD sequence variation was greater than that found in B. pseudomallei. Western blot analysis indicated that, unlike B. pseudomallei, B. thailandensis type III secreted proteins including BipD and BopE could not be detected in the supernatant of culture medium unless induced by acidic conditions. In addition, culturing B. thailandensis under acidic growth conditions (pH 4.5) can induce the ability of this bacterium to invade human respiratory epithelial cells A549. The identification of an environmental stimulus that increases the invasion capability of B. thailandensis invasion is of value for those who would like to use this bacterium as a model to study B. pseudomallei virulence.     

Macroautophagy is essential for killing of intracellular Burkholderia pseudomallei in human neutrophils

Neutrophils play a key role in the control of Burkholderia pseudomallei, the pathogen that causes melioidosis. Here, we show that survival of intracellular B. pseudomallei was significantly increased in the presence of 3-methyladenine or lysosomal cathepsin inhibitors. The LC3-flux was increased in B. pseudomallei-infected neutrophils. Concordant with this result, confocal microscopy analyses using anti-LC3 antibodies revealed that B. pseudomallei-containing phagosomes partially overlapped with LC3-positive signal at 3 and 6 h postinfection. Electron microscopic analyses of B. pseudomallei-infected neutrophils at 3 h revealed B. pseudomallei-containing phagosomes that occasionally fused with phagophores or autophagosomes. Following infection with a B. pseudomallei mutant lacking the Burkholderia secretion apparatus Bsa Type III secretion system, neither this characteristic structure nor bacterial escape into the cytosol were observed. These findings indicate that human neutrophils are able to recruit autophagic machinery adjacent to B. pseudomallei-containing phagosomes in a Type III secretion system-dependent manner.     

Burkholderia pseudomallei-induced cell fusion in U937 macrophages can be inhibited by monoclonal antibodies against host cell surface molecules

Burkholderia pseudomallei induces the formation of multinucleated giant cells in cell monolayers. After infection of human macrophage-like U937 cells with B. pseudomallei, addition of monoclonal antibodies against integrin-associated protein (CD47), E-selectin (CD62E), a fusion regulatory protein (CD98), and E-cadherin (CD324) suppressed multinucleated giant cells in a concentration-dependent manner while monoclonal antibodies against other surface molecules did not inhibit fusion despite binding to the cell surface. Flow cytometric analysis showed increased expression of CD47 and CD98, but not CD62E and CD324, upon B. pseudomallei infection. Our data suggest the involvement of specific cellular factors in the process of B. pseudomallei-induced fusion.     

Factors associated with blood zinc,chromium, and lead concentrations in residents of the Nam Pong River in Thailand

This cross-sectional analytical study aimed to determine the blood levels of zinc (B-Zn), chromium (B-Cr), and lead (B-Pb) and to identify the factors influencing these levels in the blood of residents of the Nam Pong River. Quantitative data collection was utilized, and systematic random sampling was conducted to obtain 420 samples for measuring serum heavy metals, including B-Zn, B-Cr, and B-Pb. Multiple regression analysis was used to identify factors influencing the accumulation of heavy metals in the population, reported mean differences, 95% confidence intervals, and p values. The average levels of heavy metals were 74.38 ± 14.00 µg/dL (95% confidence interval [CI]: 73.03–75.72) for zinc, 0.28 ± 0.23 µg/L (95% CI: 0.26–0.30) for chromium, and 2.80 ± 1.60 µg/dL (95% CI: 2.64–2.95) for lead, which all were within normal limits. Factors influencing zinc levels included occupational exposure (batteries) (mean diff = 11.56; 95% CI: 1.81–21.32, p value = 0.02) and consumption of fish from the river exceeding 300 grams/meal or three times/week (mean diff = 4.68; 95% CI: 0.09–9.45, p value = 0.05). Factors influencing chromium levels included a history of past illness (mean diff = 0.19; 95% CI: 0.05–0.34, p value = 0.01) and dust/chemical exposure from industry (mean diff = 0.05; 95% CI: 0.00–0.11, p value = 0.05). Factors influencing lead concentrations included gender (mean diff = 1.82; 95% CI: 0.26–1.98, p value = 0.001), smoking (mean diff = 1.03; 95% CI: 0.60–1.45, p value < 0.001), and occupational exposure (garage) (mean diff = 1.11; 95% CI: 0.27–1.94, p value = 0.01).     

Sequence- and Structure-Based Immunoreactive Epitope Discovery for Burkholderia pseudomallei Flagellin

Burkholderia pseudomallei is a Gram-negative bacterium responsible for melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management. One of the main subunit vaccine candidates is flagellin of Burkholderia pseudomallei (FliC_Bp). Here, we present the high resolution crystal structure of FliC_Bp and report the synthesis and characterization of three peptides predicted to be both B and T cell FliC_Bp epitopes, by both structure-based in silico methods, and sequence-based epitope prediction tools. All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells. Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils. The epitopes derived from this study may represent potential melioidosis vaccine components.