A novel approach to generate a recombinant toxoid vaccine against Clostridium difficile

Abstract

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.

A chimeric protein comprising the glucosyltransferase and cysteine proteinase domains of toxin B and the receptor binding domain of toxin A induces protective immunity againstClostridium difficileinfection in mice and hamsters

“…41 In addition, trans-membrane domain of TcdB has also been reported to contribute to toxicity. 42 To ensure that mTcd138 was atoxic, 2 amino acids, which have been reported to be the key residues involved in the GT activity, 43,44 were mutated in the GT domain of TcdB (Fig. 1B).…”

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

“…Kim et al reported glucosyltransferase-independent disruption of focal adhesion formation (32) and production of reactive oxygen species (33) in colonocytes induced by TcdA. Most recently, several studies have indicated that neither the CPD nor GTD enzymatic activities of TcdB are required for cellular intoxication at high toxin doses (34)(35)(36), whereas the hydrophobic region in the transloca-tion domain and GTD are important for the rapid induction of cell death (37,38). These studies utilize toxin mutagenesis, which is well known to alter protein active-site specificity or conformational integrity (39).…”

The potential for emerging therapeutic options forClostridium difficileinfection

“…A plasmid-based approach to produce a recombinant toxoid in a TcdA ¡ TcdB ¡ strain of C. difficile has also been utilised. 125 As expected, the TcdA and TcdB toxoids expressed in this plasmid system were significantly less toxic to human IMR-90 cells. Such formalin-inactivated antigens elicited a protective effect in hamster models of CDI.…”

“…Such formalin-inactivated antigens elicited a protective effect in hamster models of CDI. 125 Wang et al developed a chimeric toxin vaccine, consisting of immunogenic epitopes from both TcdA and TcdB. 126 In essence, the receptor binding domain of TcdA replaced that of TcdB and the resulting chimeric toxin displayed strong immunogenic potential.…”