Human Immune Responses to Melioidosis and Cross-Reactivity to Low-Virulence Burkholderia Species, Thailand1.

Melioidosis is a neglected tropical disease with an estimated annual mortality rate of 89,000 in 45 countries across tropical regions. The causative agent is Burkholderia pseudomallei, a gram-negative soil-dwelling bacterium. In Thailand, B. pseudomallei can be found across multiple regions, along with the low-virulence B. thailandensis and the recently discovered B. thailandensis variant (BTCV), which expresses B. pseudomallei-like capsular polysaccharide. Comprehensive studies of human immune responses to B. thailandensis variants and cross-reactivity to B. pseudomallei are not complete. We evaluated human immune responses to B. pseudomallei, B. thailandensis, and BTCV in melioidosis patients and healthy persons in B. pseudomallei-endemic areas using a range of humoral and cellular immune assays. We found immune cross-reactivity to be strong for both humoral and cellular immunity among B. pseudomallei, B. thailandensis, and BTCV. Our findings suggest that environmental exposure to low-virulence strains may build cellular immunity to B. pseudomallei

T Cell Immunity to the Alkyl Hydroperoxide Reductase of Burkholderia pseudomallei: A Correlate of Disease Outcome in Acute Melioidosis.

There is an urgent need for a better understanding of adaptive immunity to Burkholderia pseudomallei, the causative agent of melioidosis that is frequently associated with sepsis or death in patients in Southeast Asia and Northern Australia. The imperative to identify vaccine targets is driven both by the public health agenda in these regions and biological threat concerns. In several intracellular bacterial pathogens, alkyl hydroperoxidase reductases are upregulated as part of the response to host oxidative stress, and they can stimulate strong adaptive immunity. We show that alkyl hydroperoxidase reductase (AhpC) of B. pseudomallei is strongly immunogenic for T cells of 'humanized' HLA transgenic mice and seropositive human donors. Some T cell epitopes, such as p6, are able to bind diverse HLA class II heterodimers and stimulate strong T cell immunity in mice and humans. Importantly, patients with acute melioidosis who survive infection show stronger T cell responses to AhpC relative to those who do not. Although the sequence of AhpC is virtually invariant among global B. pseudomallei clinical isolates, a Cambodian isolate varies only in C-terminal truncation of the p6 T cell epitope, raising the possibility of selection by host immunity. This variant peptide is virtually unable to stimulate T cell immunity. For an infection in which there has been debate about centrality of T cell immunity in defense, these observations support a role for T cell immunity to AhpC in disease protection

HbA_1c for Melioid and Diabetes Cohorts, and treatment received.

<p>Fig 1A shows boxplots with Tukey whiskers showing the percentage glycated haemoglobin (HbA_1c) for subjects in the <i>Melioid Cohort</i> (culture-confirmed acute melioidosis) at admission to the study with no diagnosis of Diabetes Mellitus (<i>Melioid & no DM</i>), with a new diagnosis of Diabetes Mellitus defined for the study as an HbA_1c value of ≥7% (<i>Melioid & New Diagnosis DM</i>) and with known Diabetes Mellitus (<i>Melioid & Known DM</i>) alongside subjects recruited from diabetes out-patient clinic who are (<i>DM outpatient controls</i>).**** <i>P</i> < 0.0001 by Mann-Whitney test. Fig 1B shows the drug treatment for diabetes on admission for the 117 patients in the <i>Melioid Cohort</i> with previously diagnosed diabetes mellitus.</p

A nonsense mutation in <i>TLR5</i> is associated with survival and reduced IL-10 and TNF-α levels in human melioidosis

<div><p>Background</p><p>Melioidosis, caused by the flagellated bacterium <i>Burkholderia pseudomallei</i>, is a life-threatening and increasingly recognized emerging disease. Toll-like receptor (TLR) 5 is a germline-encoded pattern recognition receptor to bacterial flagellin. We evaluated the association of a nonsense <i>TLR5</i> genetic variant that truncates the receptor with clinical outcomes and with immune responses in melioidosis.</p><p>Methodology/Principal findings</p><p>We genotyped <i>TLR5</i> c.1174C>T in 194 acute melioidosis patients in Thailand. Twenty-six (13%) were genotype CT or TT. In univariable analysis, carriage of the c.1174C>T variant was associated with lower 28-day mortality (odds ratio (OR) 0.21, 95% confidence interval (CI) 0.05–0.94, <i>P</i> = 0.04) and with lower 90-day mortality (OR 0.25, 95% CI 0.07–086, <i>P</i> = 0.03). In multivariable analysis adjusting for age, sex, diabetes and renal disease, the adjusted OR for 28-day mortality in carriers of the variant was 0.24 (95% CI 0.05–1.08, <i>P</i> = 0.06); and the adjusted OR for 90-day mortality was 0.27 (95% CI 0.08–0.97, <i>P</i> = 0.04). c.1174C>T was associated with a lower rate of bacteremia (<i>P</i> = 0.04) and reduced plasma levels of IL-10 (<i>P</i> = 0.049) and TNF-α (<i>P</i> < 0.0001). We did not find an association between c.1174C>T and IFN-γ ELISPOT (T-cell) responses (<i>P</i> = 0.49), indirect haemagglutination titers or IgG antibodies to bacterial flagellin during acute melioidosis (<i>P</i> = 0.30 and 0.1, respectively).</p><p>Conclusions/Significance</p><p>This study independently confirms the association of <i>TLR5</i> c.1174C>T with protection against death in melioidosis, identifies lower bacteremia, IL-10 and TNF-α production in carriers of the variant with melioidosis, but does not demonstrate an association of the variant with acute T-cell IFN-γ response, indirect haemagglutination antibody titer, or anti-flagellin IgG antibodies.</p></div

Multivariable analysis for prediction of 28-day mortality in patients with acute melioidosis.

<p>The table displays the odds ratios for variables studied as predictors of mortality in 200 patients with acute melioidosis in a multivariable logistic regression model. Bp cell response is the log₁₀ transformed ELIspot response in fresh peripheral blood mononuclear cells to heat-killed <i>B</i>. <i>pseudomallei</i>. Neutrophil count represents the peripheral blood neutrophil count with > 4000 to 8000 neutrophils / μl as the comparator group. Odds ratios are adjusted for the other variables included in the model.</p><p>Multivariable analysis for prediction of 28-day mortality in patients with acute melioidosis.</p

Subject demographics.

<p>The gender, age and diabetes status of subjects in the study are shown. Two thirds (67%) of the patients with acute melioidosis were male, with a mean age of 55 years.</p><p>*Diabetes definitions are as follows: Pre-diagnosed = subjects with a previous diagnosis of diabetes mellitus by a doctor. Study definition = subjects with a previous diagnosis of diabetes mellitus by a doctor and / or having an HbA_1c of ≥ 7%.</p><p>Subject demographics.</p

Patients’ characteristics and mortality.

<p>Patients’ characteristics and mortality.</p