Immunogenicity of a Salmonella typhimurium aroA aroD vaccine expressing a nontoxic domain of Clostridium difficile toxin A

The C-terminal repeat domain of Clostridium difficile toxin A harbors toxin -neutralizing epitopes and is considered to be a candidate component of a v accine against C. difficile-associated disease (CDAD), Fourteen of the 38 C -terminal toxin A repeats (14CDTA) were cloned into pTECH-1 in frame with t he immunogenic fragment C of tetanus toxin (TETC) to generate plasmid p56TE TC, Expression of the TETC 14CDTA fusion protein was driven from the anaero bically inducible nirB promoter within attenuated Salmonella typhimurium BR D509 (aroA aroD), The TETC-14CDTA fusion protein was purified and shown to bind to known toxin A receptors found on the surface of rabbit erythrocytes . Intranasal (i.n.) and intragastric (i.g.) immunization with 10(7) and 10( 10) CFU, respectively, of BRD509(p56TETC) generated significant (P < 0.05) anti-toxin A serum responses after a single dose, Antibody titers were elev ated following a boosting dose with either live vaccine or a subcutaneous i njection of 0.5 mu g of purified 14CDTA protein. Importantly, serum from mi ce immunized with BRD509(p56TETC) neutralized toxin A cytotoxicity. Both i. n. and i.g. immunizations also generated toxin A-specific immunoglobulin A on the pulmonary and intestinal mucosa, respectively. Intranasal vaccinatio n induced consistently higher serum and mucosal anti-toxin A antibody respo nses. Significant anti-tetanus toroid serum and mucosal antibodies were als o generated by both immunization routes. The availability of live attenuate d Salmonella typhi for human use may allow the development of a multivalent mucosal vaccine against CDAD, tetanus, and typhoid.