Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain.

Clostridium difficile causes pseudomembranous colitis through the action of Rho-modifying proteins, toxins A and B. Antibodies directed against C. difficile toxin A prevent or limit C. difficile-induced colitis. We engineered plasmid pETR14, containing the hlyB and hlyD genes of the Escherichia coli hemolysin operon, to express a fusion protein containing 720 amino acid residues from the nontoxic, receptor-binding, carboxy terminus of C. difficile toxin A and the secretion signal of E. coli hemolysin A. We introduced pETR14 into Vibrio cholerae and found that the toxin A-HlyA fusion protein was secreted by a number of V. cholerae strains and recognized by both monoclonal and polyclonal anti-C. difficile toxin A antibodies. We introduced pETR14 into an attenuated V. cholerae strain, O395-NT, and inoculated rabbits orally with this construct. Colonization studies disclosed that the V. cholerae vector containing pETR14 was recoverable from rabbit ilea up to 5 days after oral inoculation. Vaccination produced significant systemic anti-C. difficile toxin A immunoglobulin G and anti-V. cholerae vibriocidal antibody responses. Vaccination also produced significant protection against toxin A in an ileal loop challenge assay, as assessed by determination of both fluid secretion and histological changes. These results suggest that the hemolysin system of E. coli can be used successfully in V. cholerae vector strains to effect secretion of large heterologous antigens and that a V. cholerae vector strain secreting a nontoxic, immunogenic portion of C. difficile toxin A fused to the secretion signal of E. coli HlyA induces protective systemic and mucosal immunity against this toxin.