Serum antitoxin antibodies mediate systemic and mucosal protection from Clostridium difficile disease in hamsters

Clostridium difficile is the bacterial pathogen identified as the cause of pseudomembranous colitis and is principally responsible for nosocomial antibiotic-associated diarrhea and colitis. The pathologic findings associated with this infection are believed to be caused by two large (approximately 300-kDa) exotoxins, toxins A and B. Because of the mucosal nature of this infection, vaccination strategies aimed at providing prophylactic or therapeutic immune protection have included immunization by mucosal routes. Using the hamster model of C. difficile infection, we examined the protective efficacy of inactivated toxin (toxoid) vaccine formulations prepared as either culture filtrate or partially purified toxoid. We compared combination parenteral and mucosal vaccination regimens involving intranasal, intragastric, or rectal routes of immunization and found that rectal immunization in conjunction with intramuscular (i.m.) vaccination provided full protection of hamsters from death and diarrhea while the other mucosal routes did not. Protection was associated with high levels of toxin-neutralizing antibodies in serum. The requirement for adjuvants for protection was assessed by using sequential i.m. and rectal or i.m. vaccination regimens. Unexpectedly, i.m. immunization without adjuvant conferred the highest protection from death and diarrhea; this regimen elicited the highest serum anti-toxin B titers as well as toxin B neutralizing titers. Passive transfer of mouse antitoxin antibodies protected hamsters in a dose-dependent manner, demonstrating the principal role of circulating antitoxin antibodies in immunity from this toxin-mediated mucosal disease. These results suggest that prophylactic parenteral vaccination or intravenous immunotherapy could provide protection from C. difficile disease in humans.