Glucosyltransferase-deficient Clostridium Difficile Toxins And Their Usage As Candidate Vaccines

Clostridium difficile is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans. The emergence of hypervirulent drug-resistant stains has contributed to the increased incidence and severity of C. diffiicle infection (CDI) which has become a public health concern worldwide. Antibiotic use disrupted the normal microflora in the gut, which permits C. difficile to proliferate and produce two toxins. Because of their large size and complex multifunctional domain structure, it has been a challenge to produce recombinant toxins for vaccines and immunization has thus far been limited to toxoids and peptide fragments. Here we described a novel chemeric toxin vaccine that confers complete protection against primary and recurrent CDI in mice.We have recently expressed full-length, active recombinant C. difficile toxin A and B (TcdA and TcdB) in an endotoxin-free Bacillus megaterium system. Based on this system, we generated glucosyltransferase-deficient holotoxins by point mutations and demonstrated their loss of activity in both cell and cell-free system. Two or three point mutations at substrate binding sites of GT domain did not affect functions of other domains within the toxins. These point mutations did not change the overall structure of both toxins as determined by CD scanning. The cytopathic activities, cytotoxic activities, proinflammatory acitivitis and in vivo activities of both mutant toxins and the enterotoxicity of mutant toxin A decreased dramatically as compared to wild type toxins. These results indicated that these major biological activities of TcdA and TcdB are dependent on their glucosyltransferease activities.Using mutant TcdB and formalin-inactivated TcdB (toxoid B) as antigens, we compared their immnogenecity in mice. The results showed that TcdB immunization could generate much higher IgG response and neutralizing activity. In addition, the mutant TcdA and TcdB induced potent antitoxin neutralizing antibodies showing little cross-protection. And both of these two anti-toxin neutralizing antibodies are needed for full protection against CDI.We established a conventional mouse model of CDI relapse/recurrence by treating mice with an antibiotic cocktail. Primary infection with 106 C.difficile UK1 spores caused more than 95% of the mice to develop diarrhea, with a mortality rate of around 50%. After antibiotic exposure and rechallenge of survivors with the same dose of spores one month later, mice developed relapse CDI with morbidity and mortality rates comparable to those of primary CDI. Similar symptoms were also evident in the intestines of mice that experienced the first and second episodes of CDI. Antitoxin IgG titers in sera and IgA titers in feces of mice were low before re-infection and the serum titers of neutralizing antibodies against TcdA and TcdB were barely detectable in C.difficile-infected mice. Using this model, we demonstrated that antitoxin antibodies gainst both toxins prevented recurrent CDI.To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. Immunization with the glucosyltransferase-deficient chimeric toxin cTxAB produced IgG responses and neutralizing antibodies against both TcdA and TcdB. Parenteral immunization with the avirulent toxin chimera, cTxAB, induced rapid and complete disease protection against laboratory and hypervirulent C. difficile vegetative cells and spores. Using the well-established conventional mouse model of CDI recurrence/relapse, we evaluated the protective effect of cTxAB in this model. Prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of chimeric toxins provides a novel approach for prophylactic protection of individuals at high risk of developing infectious disease such as CDI.