6 research outputs found
Immune response to recombinant Burkholderia pseudomallei FliC
Burkholderia pseudomallei is a flagellated Gram-negative bacterium which is the causative agent of melioidosis. The disease poses a major public health problem in tropical regions and diabetes is a major risk factor. The high mortality rate of melioidosis is associated with severe sepsis which involves the overwhelming production of pro-inflammatory cytokines. Bacterial flagellar protein (flagellin) activates Toll-like receptor 5 (TLR5)-mediated innate immune signaling pathways and induces adaptive immune response. However, previous studies of TLR5 signaling in melioidosis have been performed using recombinant flagellin from Salmonella Typhimurium instead of B. pseudomallei. This study aimed to investigate human innate immune response and antibody response against a recombinant B. pseudomallei flagellin (rFliC). We prepared B. pseudomallei rFliC and used it to stimulate HEK-BlueTM-hTLR5 and THP1-DualTM cells to assess TLR5 activation. Subsequently, whole blood stimulation assays with rFliC were performed ex vivo. TLR5-flagellin interactions trigger activation of transcription factor NF-κB in HEK-BlueTM-hTLR5 cells. Pro-inflammatory cytokine (IL-1β, IL-6, and TNF-α) productions from whole blood in response to rFliC differed between fourteen healthy individuals. The levels of these cytokines changed in a dose and time-dependent manner. ELISA was used to determine rFliC-specific antibodies in serum samples from different groups of melioidosis patients and healthy subjects. IgG antibody to rFliC in melioidosis patients with diabetes were higher compared with non-diabetic patients. Our results show that B. pseudomallei flagellin is a potent immune stimulator and that the immune responses to rFliC are different among individuals. This may provide valuable insights toward the potential use of rFliC in vaccine development
Immune response to recombinant Burkholderia pseudomallei FliC
Burkholderia pseudomallei is a flagellated Gram-negative bacterium which is the causative agent of melioidosis. The disease poses a major public health problem in tropical regions and diabetes is a major risk factor. The high mortality rate of melioidosis is associated with severe sepsis which involves the overwhelming production of pro-inflammatory cytokines. Bacterial flagellar protein (flagellin) activates Toll-like receptor 5 (TLR5)-mediated innate immune signaling pathways and induces adaptive immune response. However, previous studies of TLR5 signaling in melioidosis have been performed using recombinant flagellin from Salmonella Typhimurium instead of B. pseudomallei. This study aimed to investigate human innate immune response and antibody response against a recombinant B. pseudomallei flagellin (rFliC). We prepared B. pseudomallei rFliC and used it to stimulate HEK-BlueTM-hTLR5 and THP1-DualTM cells to assess TLR5 activation. Subsequently, whole blood stimulation assays with rFliC were performed ex vivo. TLR5-flagellin interactions trigger activation of transcription factor NF-κB in HEK-BlueTM-hTLR5 cells. Pro-inflammatory cytokine (IL-1β, IL-6, and TNF-α) productions from whole blood in response to rFliC differed between fourteen healthy individuals. The levels of these cytokines changed in a dose and time-dependent manner. ELISA was used to determine rFliC-specific antibodies in serum samples from different groups of melioidosis patients and healthy subjects. IgG antibody to rFliC in melioidosis patients with diabetes were higher compared with non-diabetic patients. Our results show that B. pseudomallei flagellin is a potent immune stimulator and that the immune responses to rFliC are different among individuals. This may provide valuable insights toward the potential use of rFliC in vaccine development
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Comparison of O-polysaccharide and hemolysin co-regulated protein as target antigens for serodiagnosis of melioidosis
<div><p>Background</p><p>Melioidosis is a severe disease caused by <i>Burkholderia pseudomallei</i>. Clinical manifestations are diverse and acute infections require immediate treatment with effective antibiotics. While culture is the current diagnostic standard, it is time-consuming and has low sensitivity. In endemic areas, inaccessibility to biosafety level 3 facilities and a lack of good serodiagnostic tools can impede diagnosis and disease surveillance. Recent studies have suggested that O-polysaccharide (OPS) and hemolysin co-regulated protein 1 (Hcp1) are promising target antigens for serodiagnosis of melioidosis.</p><p>Methodology/Principle findings</p><p>We evaluated rapid ELISAs using crude antigens, purified OPS and Hcp1 to measure antibody levels in three sets of sera: (i) 419 serum samples from melioidosis patients, Thai and U.S. healthy donors, (ii) 120 serum samples from patients with other bacterial infections, and (iii) 423 serum samples from 200 melioidosis patients obtained upon admission and at 12 and 52 weeks post-recovery. We observed significantly higher antibody levels using the crude antigen prepared from wild type <i>B</i>. <i>pseudomallei</i> K96243 compared to that of an OPS-mutant. The areas under receiver operator characteristics (AUROCCs) for diagnosis were compared for individual Hcp1-ELISA or OPS-ELISA or combined Hcp1/OPS-ELISA. For Thai donors, AUROCCs were highest and comparable between the Hcp1-ELISA and the combined Hcp1/OPS-ELISA (0.95 versus 0.94). For U.S. donors, the AUROCC was highest for the combined Hcp1/OPS-ELISA (0.96). Significantly higher seropositivity was observed in diabetic patients compared to those without diabetes for both the Hcp1-ELISA (87.3% versus 69.7%) and OPS-ELISA (88.1% versus 60.6%). Although antibody levels for Hcp1 were highest upon admission, the titers declined by week 52 post-recovery.</p><p>Conclusions/Significance</p><p>Hcp1 and OPS are promising candidates for serodiagnosis of melioidosis in different groups of patients. The Hcp1-ELISA performed better than the OPS-ELISA in endemic areas, thus, Hcp1 represents a promising target antigen for the development of POC tests for acute melioidosis.</p></div
Graham Bickley in uniform
Melioidosis is a fatal infectious disease caused by the environmental bacterium Burkholderia pseudomallei. It is highly endemic in Asia and northern Australia but neglected in many other tropical countries. Melioidosis patients have a wide range of clinical manifestations and definitive diagnosis requires bacterial culture which can be time-consuming. A reliable rapid serological tool is greatly needed for disease surveillance and diagnosis. We previously demonstrated by ELISA that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we have developed a rapid immunochromatography test (ICT) using Hcp1 as the target antigen (Hcp1-ICT). We evaluated this test for specific antibody detection using serum samples obtained from 4 groups of human subjects including: (i) 487 culture-confirmed melioidosis patients from four hospitals in northeast Thailand; (ii) 202 healthy donors from northeast Thailand; (iii) 90 U.S. healthy donors and (iv) 207 patients infected with other organisms. Compared to culture results as a gold standard, the sensitivity of ICT for all hospitals was 88.3%. The specificities for Thai donors and U.S. donors were 86.1% and 100%, respectively and for other infections was 91.8%. The results of the Hcp1-ICT demonstrated 92.4% agreement with the Hcp1-ELISA with kappa value of 0.829 and is much improved when compared with the current serological method, indirect hemagglutination assay (IHA) (69.5% sensitivity and 67.6% specificity for Thais). The Hcp1-ICT represents a potential point-of-care (POC) test and may be used to replace the IHA for screening of melioidosis in hospitals as well as resource-limited areas