Characteristics of the <i>Burkholderia pseudomallei</i> antigens used in this study.

<p>Characteristics of the <i>Burkholderia pseudomallei</i> antigens used in this study.</p

Development of signal intensities of grouped antigens inducing a long-term antibody response (A) and a short-term antibody response (B).

<p>Sera of individual patients (n = 36) were drawn upon admission (week 0 <i>p</i>.<i>a</i>.), 12 and 52 weeks <i>p</i>.<i>a</i>. Two graphs per antigen are shown. Left: the mean signal intensity per serum. Right: number of sera recognizing the respective antigen. Figures include only data of antigens found to be significantly recognized by melioidosis-positive sera. Statistical analyses were performed using repeated-measures ANOVA followed by Bonferroni's Multiple Comparison test comparing signal intensities measured in sera of week 0, 12 and 52 <i>p</i>.<i>a</i>. (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001)</p

Comparative calculation of IHA and protein array sensitivity.^#

<p>Comparative calculation of IHA and protein array sensitivity.<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004847#t003fn001" target="_blank">^#</a></p

Rapid and Sensitive Multiplex Detection of <i>Burkholderia pseudomallei</i>-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach

<div><p>Background</p><p>The environmental bacterium <i>Burkholderia pseudomallei</i> causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against <i>B</i>. <i>pseudomallei</i>, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against <i>B</i>. <i>pseudomallei</i> is still lacking.</p><p>Methods and Principal Findings</p><p>In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified <i>B</i>. <i>pseudomallei</i> proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; <i>p</i> = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response.</p><p>Conclusions</p><p>Our protein array provides a standardized, rapid, easy-to-perform test for the detection of <i>B</i>. <i>pseudomallei</i>-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-<i>B</i>. <i>pseudomallei</i> antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.</p></div

Experimental timeline of probing a collection of positive sera drawn from single melioidosis patients (n = 36) upon admission (week 0) and after 12 and 52 weeks <i>p</i>.<i>a</i>.

<p>All sera were sampled in Ubon Ratchathani, Thailand. The antigens are shown in rows with five increasing concentrations per protein, and the patient samples are represented in columns. Array signals are reflected by the intensities of the color (white to blue) inside the boxes. The heatmap was created using Multi experiment Viewer (MeV 4.9.0) from TM4 suite, USA.</p

Construction of a <i>B</i>. <i>pseudomallei</i> protein microarray.

<p>The protein array was spotted with 20 different <i>B</i>. <i>pseudomallei</i> proteins and further internal positive and negative controls, for a total of 445 protein spots. All proteins were applied in triplicate and at five dilutions (0.01 mg/ml to 0.45 mg/ml) on glass slides, including human IgG and IgM controls and further other internal controls, i.e., murine IgG and IgM, bovine IgG, porcine IgG, caprine IgG and ovine IgG controls. All protein arrays were incubated <b>A</b> only with anti-human-IgG antibodies (empty control), <b>B</b> with melioidosis-positive or <b>C</b> melioidosis-negative blood sera or blood plasmas at 1:1000 dilutions (<b>A, B</b> and <b>C</b> are representative images). IgG antibodies bound to <i>B</i>. <i>pseudomallei</i> antigens were detected using horseradish-peroxidase (HRP) linked secondary anti-human-IgG antibodies and 3,3’,5,5’-tetramethyl-benzidine (TMB), which caused a blue precipitate. Protein arrays were read out by the ArrayMate (Alere Technologies GmbH, Germany). Highlighted are human IgG controls (green rectangle) and horseradish-peroxidase controls (red rectangle); all other controls are not shown.</p

Average signal intensities of IgG antibodies bound to <i>B</i>. <i>pseudomallei</i> proteins probed with melioidosis-positive and negative control samples.

<p>The diagram shows the average signal intensity of each antigen (spotted protein solution of 0.45 mg/ml) incubated with sera from melioidosis-positive groups (week 0, 12 and 52 <i>p</i>.<i>a</i>.), healthy control individuals from endemic and non-endemic areas, as well as samples from patients with other bacteremia or fungaemia obtained in the non-endemic area of Greifswald. Not shown are values for antigens with His-tag. Error bars indicate standard error of the mean (SEM).</p

Measured IHA titers (A) and protein-array–derived average signal intensities (B) per serum.

<p>Melioidosis-positive and -negative samples were drawn in endemic areas of Thailand. Additionally, means with standard error of the mean (SEM) are shown for each group. The IHA titer was determined using the indirect hemagglutionation assay described elsewhere (<a href="http://www.melioidosis.info/home.aspx" target="_blank">http://www.melioidosis.info/home.aspx</a>). Statistical analyses were performed using the Kruskal-Wallis test followed by Dunn's Multiple Comparison test, comparing titers or signal intensities measured in sera of weeks 0 (n = 75), 12 (n = 50), and 52 (n = 46), as well as from healthy individuals (n = 100) (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001).</p

Heatmap of probing a collection of melioidosis-positive sera and negative control sera.

<p>Protein arrays containing 20 <i>B</i>. <i>pseudomallei</i> recombinant proteins were probed with 260 melioidosis and nonmelioidosis sera. The melioidosis positive sera (n = 75) were drawn at week 0 (<i>p</i>.<i>a</i>.) from patients with <i>B</i>. <i>pseudomallei</i> infections. All positive sera were sampled in Ubon Ratchathani, Thailand. Negative control sera of healthy persons (n = 125) were sampled in the endemic regions of Thailand ((Ubon Ratchathani (U.R.) and Bangkok (B.)), Thailand and non-endemic region of Greifswald, Germany. Additionally, further negative control sera of patients with other bacteremia or fungaemia (n = 60) were used from the non-endemic region of Greifswald. Not shown are the results of incubations with meliodosis-positive sera obtained 12 and 52 weeks after admission. The antigens are shown in rows with five increasing concentrations per protein, and the patient samples are represented in columns. Array signals are reflected by the intensities of the color (white to blue) inside the boxes. The heatmap was created using Multi experiment Viewer (MeV 4.9.0) from TM4 suite, USA.</p

Table_4_Deciphering the human antibody response against Burkholderia pseudomallei during melioidosis using a comprehensive immunoproteome approach.xlsx

IntroductionThe environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients.Methods and resultsIn this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio.ConclusionOur study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.</p