The 20 proteins that showed the greatest difference in response between these groups (lowest values) are shown in Figure 3

The 20 proteins that showed the greatest difference in response between these groups (lowest values) are shown in Figure 3. episodes of clinical illness, with 6%C13% of these cases due to reinfection but the majority a consequence of the failure to eliminate the pathogen after treatment with antimicrobials [5]. The host immune responses required to recover from melioidosis or to prevent infection in humans living in melioidosis-endemic areas are largely unknown. With use of a murine model of melioidosis, both cell-mediated and humoral immune responses have been shown to play roles in protection [6]. Cell-mediated responses involving natural killer (NK) cells and adaptive T cells producing interferon- (IFN-) play an important role in control of infection [7C9]. Our previous studies have revealed that memory CD4+, CD8+T Ulipristal acetate (TEMRA), and NK cells from seropositive healthy individuals living in endemic areas or from individuals who have recovered from melioidosis are primed and produce IFN- in vitro in response to killed or the bacterial ABC transporter proteins, LolC, OppA, or PotF. The magnitude of these cellular responses correlated with antibody titers to killed cells detected by means of conventional indirect hemagglutination assay (IHA) [10]. However, the identity of other antigens recognized by the plasma of these individuals is not known. High-throughput protein microarrays have previously been developed and used to map the humoral responses to individual bacterial and viral proteins [11C16]. Recently, we have devised a protein array and probed it with serum specimens from acute melioidosis patients in Northeast Thailand and Singapore. Mapping the profile of antibody responses has allowed the identification of proteins that can be used as serodiagnostic antigens for melioidosis [17]. The potential for these antigens to stimulate cell-mediated immune responses and the Ulipristal acetate identification of IL22RA1 proteins that could induce protective immune responses has not been reported. This study aimed to identify proteins that could be candidate protective antigens. A protein array Ulipristal acetate was probed with plasma from individuals who had recovered from melioidosis after receiving antibiotic therapy and from seropositive individuals living in endemic areas but with no history of melioidosis. We also sought to determine whether recurrent disease, septic disease, or localized infection influenced the antibody response profile and how these antibody responses were related to T cell responses in individuals. In the longer term, our results will support research to devise vaccines against melioidosis. MATERIALS AND METHODS Blood Samples Recovered melioidosis patients and healthy control individuals were enrolled in this study and recruited by a study team based at Sappasithiprasong Hospital, Ubon Ratchathani, Northeast Thailand. Ethical permission was obtained from Ethical KKU research, no. “type”:”entrez-nucleotide”,”attrs”:”text”:”HE470506″,”term_id”:”288761517″,”term_text”:”HE470506″HE470506 (Scanning the proteome for vaccine antigens). Recovered melioidosis patients were defined as individuals who had a history of clinical melioidosis (confirmed by culture positive for from clinical samples) but at the time of blood collection had completed a course of antibiotic treatment and had no sign of active melioidosis. Recurrent melioidosis infection was defined as new symptoms and signs of infection in association with a culture positive for following previous treatment and response to oral antibiotic therapy [5]. Healthy control individuals had no history of melioidosis and included seropositive individuals tested by means of IHA (titer, 40) and seronegative individuals. Plasma samples from 72 recovered melioidosis patients and 108 control individuals were used to probe protein arrays, and blood samples from 30 recovered melioidosis patients and 20 healthy control individuals were used for cell-mediated immune response assays. The details of sample demographic characteristics have been described elsewhere [10]. Antibody Detection with Use of Protein Microarray Analysis Fabrication of the protein array and probing with plasma samples were as described elsewhere [18]. Ulipristal acetate In brief, strain K96243 DNA was used as a template for the polymerase chain reaction (PCR). PCR products were cloned into a T7 expression vector by means of homologous recombination. Purified plasmids were expressed in the antigens that were downselected from a larger 1205 proteome array after probing with melioidosis patient serum [17]. In addition, 4 serial dilutions of human IgG, 6 spots comprising RTS-100 reactions with nonrecombinant template vector, and 4 serial dilutions of EBNA1 protein were also printed as internal positive, negative, and serum controls, respectively. Plasma was reacted with the chips using a previously reported protocol [18]. Briefly, the 16-pad Bp chip was prewetted with 80 L of blocking buffer (Whatman) at 25oC for Ulipristal acetate 30 min. Serum samples were diluted 1:50 in blocking buffer containing 10% lysate and incubated for 2 h at 25oC with orbital shaking at 90 rpm. The arrays were washed 7 times.

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