Development of an Antitoxin Vaccine Against Enterotoxigenic Esherichia coli Mediated Diarrheal Disease

Enterotoxigenic E. coli (ETEC) causes significant morbidity and mortality in both humans and animals due to diarrheal disease. Many virulence factors of bacterial pathogenesis have been well characterized and vaccine studies have been conducted to target the most prevalent virulent determinants circulating in the field. Following the colonization at host small intestine, an ETEC strain produces at least one of the two types of enterotoxins: heat-stable toxin (ST) and heat-labile toxin (LT), two critical virulence factors associated with diarrheal disease. In order to use ST and LT as vaccine components, they need to be detoxified to minimize side effects related to their toxicity. In addition, due to the poor immunogenicity, ST should be linked to a carrier protein to induce immune responses. In this study, we constructed a toxoid fusion protein 3x mSTatmLT with three copies of a STa toxoid (STa A14Q), and one copy of an LT toxoid (LTS63K/R192G/ L211A) to used it as an antigen. The fusion protein was shown to be safe as it has little effects on the intracellular cyclic GMP and cyclic AMP levels of T84 cell line. The immunogenicity of the fusion protein was tested in BALB/c mice by intraperitoneal (IP), intranasal (IN), and sublingual (SL) immunization routes. Anti-STa antibodies were detected in serum and intestine wash samples in mice immunized by intraperitoneal and intranasal routes, and in intestine wash samples in mice immunized by sublingual route. Anti-LT antibodies were also detected in serum and intestine wash samples in mice vaccinated through intraperitoneal, intranasal, and sublingual routes, and in fecal samples in mice vaccinated through intranasal routes. All anti-LT antibodies were shown to neutralize cholera toxin (CT) in vitro, a highly homologous toxin to LT, and some anti- STa antibodies were shown to neutralize STa in vitro. This study demonstrates that the detoxified 3x mSTa-tmLT fusion protein is safe and immunogenic and it can elicit neutralizing antibodies. The fusion protein has the potential to serve as a vaccine candidate to stimulate the anti-toxin immunity against ETEC strains producing LT and/or STa