Recombinant unpurified rETX(H106P)/CTB-rETX(Y196E) protects rabbits against Clostridium perfringens epsilon toxin

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, has been touted as a potential biological weapon and is known to induce fatal enterotoxemia in a variety of livestock animals. For the efficient production of recombinant proteins with the objective of investigating the effects of different recombinant vaccines against ETX, a bicistronic design (BCD) expression system including the ETX coding sequence with mutation of amino acid 106 from Histidine to Proline (ETX(H106P)) in the first cistron, followed by Cholera Toxin B (CTB) linked with the ETX coding sequence with mutation of amino acid 196 from Tyrosine to Glutamic acid (ETX(Y196E)) in the second cistron, was generated under the control of a single promoter. Rabbits were immunized twice with five inactivated recombinant Escherichia coli (E. coli) vaccines containing 100 µg/ml of the recombinant mutant rETX(H106P)/CTB-rETX(Y196E) proteins mixed with different adjuvants. Apart from rETX(H106P)/CTB-rETX(Y196E)-IMS1313-vaccinated rabbits, the neutralizing antibody titers of rETX(H106P)/CTB-rETX(Y196E)-vaccinated rabbits were higher after the initial immunization than those administered the ETX toxoid or current commercial vaccines. rETX(H106P)/CTB-rETX(Y196E) mixed with ISA201 induced the highest neutralizing antibody titer of 120 after the first immunization, suggesting that 0.1 ml of pooled sera could neutralize 120× mouse LD(100) (100% lethal dose) of ETX. Following the second vaccination, rETX(H106P)/CTB-rETX(Y196E) mixed with ISA201 or GR208 produced the highest neutralizing titer of 800. Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD(100) of ETX challenge. These results show that these novel recombinant proteins can induce a strong immune response and represent potential targets for the development of a commercial vaccine against the C. perfringens epsilon toxin.