Burkholderia pseudomallei causes melioidosis, a common source of pneumonia and sepsis in Southeast Asia and Northern Australia that results in high mortality rates. A caprine melioidosis model of aerosol infection that leads to a systemic infection has the potential to characterize the humoral immune response. This could help identify immunogenic proteins for new diagnostics and vaccine candidates. Outbred goats may more accurately mimic human infection, in contrast to the inbred mouse models used to date. B. pseudomallei infection was delivered as an intratracheal aerosol. Antigenic protein profiling was generated from the infecting strain MSHR511. Humoral immune responses were analyzed by ELISA and western blot, and the antigenic proteins were identified by mass spectrometry. Throughout the course of the infection the assay results demonstrated a much greater humoral response with IgG antibodies, in both breadth and quantity, compared to IgM antibodies. Pre-infection sera showed multiple immunogenic proteins already reactive for IgG (7–20) and IgM (0–12) in most of the goats despite no previous exposure to B. pseudomallei. After infection, the number of IgG reactive proteins showed a marked increase as the disease progressed. Early stage infection (day 7) showed immune reaction to chaperone proteins (GroEL, EF-Tu, and DnaK). These three proteins were detected in all serum samples after infection, with GroEL immunogenically dominant. Seven common reactive antigens were selected for further analysis using ELISA. The heat shock protein GroEL1 elicited the strongest goat antibody immune response compared to the other six antigens. Most of the six antigens showed the peak IgM reactivity at day 14, whereas the IgG reactivity increased further as the disease progressed. An overall MSHR511 proteomic comparison between the goat model and human sera showed that many immune reactive proteins are common between humans and goats with melioidosis.

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative soil bacterium well recognized in Southeast Asia and northern Australia. However, wider and expanding global distribution of B. pseudomallei has been elucidated. Early diagnosis is critical for commencing the specific therapy required to optimize outcome. Serological testing using the indirect hemagglutination (IHA) antibody assay has long been used to augment diagnosis of melioidosis and to monitor progress. However, cross reactivity and prior exposure may complicate the diagnosis of current clinical disease (melioidosis). The goal of our study was to develop and initially evaluate a serology assay (BurkPx) that capitalized upon host response to multiple antigens. Antigens were selected from previous studies for expression/purification and conjugation to microspheres for multiantigen analysis. Selected serum samples from non-melioidosis controls and serial samples from culture-confirmed melioidosis patients were used to characterize the diagnostic power of individual and combined antigens at two times post admission. Multiple variable models were developed to evaluate multivariate antigen reactivity, identify important antigens, and determine sensitivity and specificity for the diagnosis of melioidosis. The final multiplex assay had a diagnostic sensitivity of 90% and specificity of 93%, which was superior to any single antigen in side-by-side comparisons. The sensitivity of the assay started at >85% for the initial serum sample after admission and increased to 94% 21 days later. Weighting antigen contribution to each model indicated that certain antigen contributed to diagnosis more than others, which suggests that the number of antigens in the assay can be decreased. In summation, the BurkPx assay can facilitate the diagnosis of melioidosis and potentially improve on currently available serology assays. Further evaluation is now required in both melioidosis-endemic and non-endemic settings.

29Burkholderia pseudomallei causes melioidosis, a common source of pneumonia and sepsis in 30 Southeast Asia and Northern Australia, that results in high mortality rates. A caprine melioidosis 31 model of aerosol infection that leads to a systemic infection has the potential to characterize the 32 humoral immune response. This could help identify immunogenic proteins for new diagnostics 33 and vaccine candidates. Outbred goats may more accurately mimic human infection, in contrast to 34 the inbred mouse models used to date. B. pseudomallei infection was delivered as an intratracheal 35 aerosol. Antigenic protein profiling was generated from the infecting strain MSHR511. Humoral 36 immune responses were analyzed by ELISA and western blot, and the antigenic proteins were 37 identified by mass spectrometry. Throughout the course of the infection the assay results 38 demonstrated a much greater humoral response with IgG antibodies, in both breadth and quantity, 39 compared to IgM antibodies. Pre-infection sera showed multiple immunogenic proteins already 40 reactive for IgG (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and IgM (0-12) in most of the goats despite no previous exposure to B. 41 pseudomallei. After infection, the number of IgG reactive proteins showed a marked increase as 42 the disease progressed. Early stage infection (day 7) showed immune reaction to chaperone 43 proteins (GroEL, EF-Tu, and DnaK). These three proteins were detected in all serum samples after 3 44 infection, with GroEL immunogenically dominant. Seven common reactive antigens were selected 45 for further analysis using ELISA. The heat shock protein GroEL1 elicited the strongest goat 46 antibody immune response compared to the other six antigens. Most of the six antigens showed 47 the peak IgM reactivity at day 14, whereas the IgG reactivity increased further as the disease 48 progressed. An overall MSHR511 proteomic comparison between the goat model and human sera 49 showed that many immune reactive proteins are common between humans and goats with 50 melioidosis. 51 52 Author Summary 53 B. pseudomallei infection, the causative agent of melioidosis, results in severe 54 disseminated or localized infections. A systemic study of the humoral immune response to B. 55 pseudomallei infection using the B. pseudomallei aerosol caprine model would help understand 56 the detectable antigenic proteins as the infection progresses. To study the immune response, IgG 57 and IgM antibody responses to whole cell lysate proteins were identified and analyzed. Antigenic 58 carbohydrates were also studied. From the results, this study suggests that the caprine humoral 59 immune response to aerosolized B. pseudomallei has similarities to human melioidosis and may 60 facilitate the analysis of the temporal antibody responses. In addition, commonly detected 61 immunogenic proteins may be used as biomarkers for the future point of care (POC) diagnostics.62 4 63 Introduction 64 Burkholderia pseudomallei is a Gram-negative, non-spore formin...

Background Melioidosis is a disease caused by the bacterium Burkholderia pseudomallei, infecting humans and non-human primates (NHP) through contaminated soil or water. World-wide there are an estimated 165,000 human melioidosis cases each year, but recordings of NHP cases are sporadic. Clinical detection of melioidosis in humans is primarily by culturing B. pseudomallei, and there are no standardized detection protocols for NHP. NHP are an important animal model for melioidosis research including clinical trials and development of biodefense countermeasures. Methodology/Principle findings We evaluated the diagnostic potential of the multiple antigen serological assay, BurkPx, in NHP using two sera sets: (i) 115 B. pseudomallei-challenged serum samples from 80 NHP collected each week post-exposure (n = 52) and at euthanasia (n = 47), and (ii) 126 B. pseudomallei-naïve/negative serum samples. We observed early IgM antibody responses to carbohydrate antigens followed by IgG antibody recognition to multiple B. pseudomallei protein antigens during the second week of infection. B. pseudomallei negative serum samples had low to intermediate antibody cross reactivity to the antigens in this assay. Infection time was predicted as the determining factor in the variation of antibody responses, with 77.67% of variation explained by the first component of the principal component analysis. A multiple antigen model generated a binary prediction metric (p^), which when applied to all data resulted in 100% specificity and 63.48% sensitivity. Removal of week 1 B. pseudomallei challenged serum samples increased the sensitivity of the model to 95%. Conclusion/Significance We employed a previously standardized assay for humans, the BurkPx assay, and assessed its diagnostic potential for detection of B. pseudomallei exposure in NHP. The assay is expected to be useful for surveillance in NHP colonies, in investigations of suspected accidental releases or exposures, and for identifying vaccine correlates of protection.