Clostridium perfringens epsilon prototoxin mutant rpETXY30A/Y71A/H106P/Y196A as a vaccine candidate against enterotoxemia

Epsilon toxin (ETX) is secreted by Clostridium perfringens (C. perfringens) as a relatively inactive prototoxin (pETX), which is enzymatically activated to ETX by removing carboxy-terminal and amino-terminal peptides. Genetically engineered ETX mutants have been shown to function as potential vaccine candidates in the prevention of the enterotoxemia caused by C. perfringens. In the present study, two recombinant site-directed mutants of pETX, rpETXY30A/Y71A/H106P/Y196A (rpETXm41) and rpETXY30A/H106P/Y196A/F199E (rpETXm42), were synthesized by mutating four essential amino acid residues (Tyr30, Tyr71, His106, Tyr196 or Phe199). Compared to recombinant pETX (rpETX), both rpETXm41 and rpETXm42 lacked the detectable toxicity in MDCK cells and mice, which suggested that both rpETXm41 and rpETXm42 are sufficiently safe to be vaccine candidates. Despite the fact that rpETXm41 and rpETXm42 were reactogenic with polyclonal antibodies against crude ETX, both single- and double-dose vaccination (Vs and Vd, respectively) of rpETXm41 induced a higher level of IgG titer and protection in mice than that of rpETXm42. Therefore, we selected rpETXm41 for the further study. Sheep received Vs of 150 μg rpETXm41 developed significant levels of toxin-neutralizing antibodies persisting for at least 6 months, which conferred protection against crude ETX challenge without microscopic lesions. These data suggest that genetically detoxified rpETXY30A/Y71A/H106P/Y196A could form the basis of a next-generation enterotoxemia vaccine.