Evaluation of the immunogenicity and protective efficacy of a recombinant CS6-based ETEC vaccine in an Aotus nancymaae CS6 + ETEC challenge model.

Colonization factors or Coli surface antigens (CFs or CS) are important virulence factors of Enterotoxigenic E. coli (ETEC) that mediate intestinal colonization and accordingly are targets of vaccine development efforts. CS6 is a highly prevalent CF associated with symptomatic ETEC infection both in endemic populations and amongst travelers. In this study, we used an Aotus nancymaae non-human primate ETEC challenge model with a CS6 + ETEC strain, B7A, to test the immunogenicity and protective efficacy (PE) of a recombinant CS6-based subunit vaccine. Specifically, we determined the ability of dscCssBA, the donor strand complemented recombinant stabilized fusion of the two subunits of the CS6 fimbriae, CssA and CssB, to elicit protection against CS6 + ETEC mediated diarrhea when given intradermally (ID) with the genetically attenuated double mutant heat-labile enterotoxin LT(R192G/L211A) (dmLT). ID vaccination with dscCssBA + dmLT induced strong serum antibody responses against CS6 and LT. Importantly, vaccination with dscCssBA + dmLT resulted in no observed diarrheal disease (PE = 100%, p = 0.03) following B7A challenge as compared to PBS immunized animals, with an attack rate of 62.5%. These data demonstrate the potential role that CS6 may play in ETEC infection and that recombinant dscCssBA antigen can provide protection against challenge with the homologous CS6 + ETEC strain, B7A, in the Aotus nancymaae diarrheal challenge model. Combined, these data indicate that CS6, and more specifically, a recombinant engineered derivative should be considered for further clinical development.

Oral delivery of Hyperimmune bovine serum antibodies against CS6-expressing enterotoxigenic Escherichia coli as a prophylactic against diarrhea.

BACKGROUND: . Oral administration of bovine antibodies active against enterotoxigenic Escherichia coli (ETEC) have demonstrated safety and efficacy against diarrhea in human challenge trials. The efficacy of bovine serum immunoglobulins (BSIgG) against recombinant colonization factor CS6 or whole cell ETEC strain B7A was assessed against challenge with the CS6-expressing B7A. METHODS: . This was a randomized, double-blind, placebo-controlled trial in which healthy adults received oral hyperimmune BSIgG anti-CS6, anti-B7A whole cell killed or non-hyperimmune BSIgG (placebo) in a 1:1:1 ratio then challenged with ETEC B7A. Two days pre-challenge, volunteers began a thrice daily, seven day course of immunoprophylaxis. On day 3, subjects received 1 × 1010 CFUs of B7A. Subjects were observed for safety and the primary endpoint of moderate-severe diarrhea (MSD). RESULTS: . A total of 59 volunteers received product and underwent ETEC challenge. The BSIgG products were well-tolerated across all subjects. Upon challenge, 14/20 (70%) placebo recipients developed MSD, compared to 12/19 (63%; p = .74) receiving anti-CS6 BSIgG and 7/20 (35%; p = .06) receiving anti-B7A BSIgG. Immune responses to the ETEC infection were modest across all groups. CONCLUSIONS: . Bovine-derived serum antibodies appear safe and well tolerated. Antibodies derived from cattle immunized with whole cell B7A provided 50% protection against MSD following B7A challenge; however, no protection was observed in subjects receiving serum antibodies targeting CS6. The lack of observed efficacy in this group may be due to low CS6 surface expression on B7A, the high dose challenge inoculum and/or the use of serum derived antibodies versus colostrum-derived antibodies.

The novel sigma54- and sigma28-dependent flagellar gene transcription hierarchy of Vibrio cholerae.

The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum. Flagellar transcriptional regulatory factors have been demonstrated to contribute to V. cholerae virulence, but the role these factors play in the transcription hierarchy controlling flagellar synthesis has been unclear. The flagellar genes revealed by the V. cholerae genome sequence are located in three large clusters, with the exception of the motor genes, which are found in three additional locations. It had previously been demonstrated that the alternative sigma factor sigma54 and the sigma54-dependent activators FlrA and FlrC are necessary for flagellar synthesis. The V. cholerae genome sequence revealed the presence of a fliA gene, which is predicted to encode the alternative flagellar sigma factor sigma28. A V. cholerae DeltafliA mutant strain is non-motile, and synthesizes a truncated flagellum. Vibrio cholerae FliA complements both V. cholerae and Salmonella typhimurium fliA mutants for motility, consistent with its function as an alternative flagellar sigma factor. Analysis of lacZ transcriptional fusions of the V. cholerae flagellar promoters in both V. cholerae and S. typhimurium identified sigma28-, sigma54-, FlrA- and FlrC-dependent promoters, as well as promoters that were independent of all these factors. Our results support a model of V. cholerae flagellar gene transcription as a novel hierarchy composed of four classes of genes. Class I is composed solely of the gene encoding the sigma54-dependent activator FlrA, which along with the sigma54-holoenzyme form of RNA polymerase activates expression of Class II genes. These genes include structural components of the MS ring, switch and export apparatus, as well as the genes encoding both FliA and FlrC. FlrC, along with sigma54-holoenzyme, activates expression of Class III genes, which include basal body, hook and filament genes. Finally, sigma28-holoenzyme activates expression of Class IV genes, which include additional filament genes as well as motor genes. Thus, this novel V. cholerae flagellar hierarchy has incorporated elements from both the sigma54-dependent Caulobacter crescentus polar flagellar hierarchy and the sigma28-dependent S. typhimurium peritrichous flagellar hierarchy.