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BackgroundCholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera.MethodsWe probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay.ResultsOverall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O-specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate-2-oxoglutarate aminotransferase.ConclusionsThis study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine.

Epitope-based vaccines permit the selection of only a specific subset of epitopes to induce the necessary immune response, thus providing a rational alternative to conventional design approaches. Using a range of immunoinformatics tools, we identified a novel, contiguous 28 amino acid multi-epitope cluster within the highly conserved secretory protein Ag85B of Mycobacterium tuberculosis, the causative agent of TB. This cluster, named Ep85B, is composed of epitopes which bind to three HLA Class I and 15 Class II molecules, and harbors the potential to generate 99% population coverage in TB-endemic regions. We experimentally evaluated the capacity of Ep85B to elicit T-cell immune responses using whole blood cells and, as predicted, observed significant increases in populations of both CD4+ and memory CD4+ CD45RO+ T-cells. Our results demonstrate the practical utility of an epitope-based design methodology - a strategy that, following further evaluation, may serve as an additional tool for the development of novel vaccine candidates against TB and other diseases., AuthorCount:10

Patients with acute watery diarrhea caused by Vibrio cholerae O1 or enterotoxigenic Escherichia coli (ETEC) were analyzed for innate immune factors produced by the epithelium during the disease process. Duodenal biopsies were obtained from study participants at the acute (day 2) and convalescent (day 21) stages of disease. Levels of alpha-defensin (HD-5 and -6), beta-defensin (hBD-1-4), and cathelicidin (LL-37) mRNAs were determined by real-time qRT-PCR. hBD-2, HD-5, LL-37 peptides were analyzed in duodenal epithelium by immunomorphometry. Concentration of hBD-2 in stool was determined by ELISA. Specimens from healthy controls were also analyzed. hBD-2 mRNA levels were significantly increased at acute stage of diarrhea; hBD-2 peptide was detected in fecal specimens but barely in duodenal epithelium at acute stage. Immunomorphometry analysis showed that Paneth cells contain significantly higher amounts of HD-5 pre/propeptide at convalescence (P < 0.01) and in healthy controls (P < 0.001) compared to acute stage, LL-37 peptide levels also decreased at acute stage while mRNA levels remained unchanged. mRNA expression levels of the other antimicrobial peptides remained unchanged with higher levels of alpha-defensins than beta-defensins. V cholerae induced an innate immune response at the acute stage of disease characterized by increased expression of hBD-2, and continued expression of hBD-1, HD-5-6, and LL-37.