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 acti on of Rho-modifying proteins, toxins A and B. Antibodies directed agai nst 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 con taining 720 amino acid residues from the nontoxic, receptor-binding, c arboxy 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. c holerae strains and recognized by both monoclonal and polyclonal anti- C. difficile toxin A antibodies. We introduced pETR14 into an attenuat ed V. cholerae strain, O395-NT, and inoculated rabbits orally with thi s construct. Colonization studies disclosed that the V. cholerae vecto r containing pETR14 was recoverable from rabbit ilea up to 5 days afte r oral inoculation. Vaccination produced significant systemic anti-C. difficile toxin A immunoglobulin G and anti-V. cholerae vibriocidal an tibody responses. Vaccination also produced significant protection aga inst toxin A in an ileal loop challenge assay, as assessed by determin ation of both fluid secretion and histological changes. These results suggest that the hemolysin system off. coli can be used successfully i n V. cholerae vector strains to effect secretion of large heterologous antigens and that a V. cholerae vector strain secreting a nontoxic, i mmunogenic portion of C. difficile toxin A fused to the secretion sign al of E. coli HlyA induces protective systemic and mucosal immunity ag ainst this toxin.