C. difficile Toxoid Vaccine(Sanofi Pasteur)

Clostridium difficile infection (CDI) has been identified as the leading cause of nosocomial diarrhoea and pseudomembranous colitis associated with antibiotic therapy. Recent epidemiological changes as well as increases in the number of outbreaks of strains associated with increased virulence and higher mortality rates underscore the importance of identifying alternatives to antibiotics to manage this important disease. Animal studies have clearly demonstrated the roles that toxins A and B play in gut inflammation as well as diarrhoea; therefore it is not surprising that serum anti-toxin A and B IgG are associated with protection against recurrent CDI. In humans, strong humoral toxin-specific immune responses elicited by natural C. difficile infection is associated with recovery and lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. The first generation of C. difficile vaccine that contained inactivated toxin A and B was found to be completely protective against death and diarrhoea in the hamster C. difficile challenge model. When tested in young healthy volunteers in Phase I clinical trials, this investigational vaccine was shown to be safe and immunogenic. Moreover, in a separate study this vaccine was able to prevent further relapses in three out of three patients who had previously suffered from chronic relapsing C. difficile-associated diarrhoea. Herein we examined the immunogenicity and protective activity of a next-generation Sanofi Pasteur two-component highly purified toxoid vaccine in a C. difficile hamster model. This model is widely recognized as a stringent and relevant choice for the evaluation of novel treatment strategies against C. difficile and was used in preclinical testing of the first-generation vaccine candidate. Intramuscular (i.m.) immunizations with increasing doses of this adjuvanted toxoid vaccine protected hamsters from mortality and disease symptoms in a dose-dependent manner. ELISA measurements of pre-challenge sera showed that the median anti-toxin A and anti-toxin B IgG titres in the group of surviving animals were significantly higher than the median values in the group of animals that did not survive challenge. Assessment of the neutralizing activity of these sera revealed a statistically significant difference between the levels of both toxin A and toxin B neutralizing titres in protected versus unprotected animals as the median anti-toxin A and anti-toxin B neutralizing titres from surviving animals were higher than the median values from animals that succumbed to challenge. Statistically significant correlations between the toxin-specific binding titres and toxin neutralizing titres were seen for both toxin A and toxin B responses. The role of circulating anti-toxin antibodies in immunity against disease was evaluated by passive transfer of immune sera against C. difficile toxoids to naïve hamsters. Passively immunized animals were protected against morbidity and mortality associated with C. difficile challenge. Taken together, these results indicate the ability of i.m. immunization with inactivated toxins A and B to induce robust dose-dependent anti-toxin A and anti-toxin B IgG responses, the principal role of circulating anti-toxin antibody in immunity against disease and that antibody toxin binding and neutralization titres can serve as correlates of protection in the hamster challenge model of C. difficile.

The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.