Synthesis of peptide based epsilon toxin vaccine by covalent anchoring to tetanus toxoid

Abstract

The epsilon toxin (Etx) produced by Clostridium perfringens type B and D causes severe enterotoxaemia associated with a general edema and neurological alterations, leading to subsequent death and is listed as one of the most lethal toxins. Currently employed vaccines against C. perfringens epsilon toxin include toxoid based vaccines. Use of peptide vaccines has become an interesting approach for vaccination after the successful licensing of peptide vaccines against Haemophilus influenza, Neisseria meningitides and Streptococcus pneumonia that have demonstrated the potential and effectiveness of these vaccines. Therefore, the present study was undertaken to develop a peptide based vaccine against epsilon toxin. Peptides were selected on the basis of epitope mapping by making 35 overlapping peptides of 15 amino acid residues in length specific to the primary amino acid sequence of the toxin, with a 7 amino acid residues overlaps between sequential peptides. Chemically synthesized peptides that were recognised by the antibody against the full length epsilon toxin were further assessed for vaccine potential. The selected peptides were chemically conjugated to partially reduced tetanus toxoid (TT) using of N-succinimidyl-3(2-pyridyldithio) propionate. Immunization of BALB/c mice with TT-peptide conjugates by sub-cutaneous route induced sustained high level mixed immune response as analyzed by antibody isotyping. Immunoblot analysis and ELISA clearly indicated generation of Etx-specific antibodies. Further, neutralization studies with the antisera generated against the TT-conjugated peptide(s) demonstrated that the antisera were able to neutralize the lethal dose of epsilon toxin in vitro demonstrating its potential as a promising vaccine candidate against enterotoxaemia.