Studies on the immunogenicity and stability of the epsilon prototoxin of Clostridium perfringens type D detoxified by controlled iodination

PARREIRA, M.P., AURÉLIO, R., CAMPOS, P.C., JÚNIOR, A.D.C., LOBATO, A, F.C.F., HENEINE, L.G.D.

ImmunologyLaboratory, Fundação Ezequiel Dias, Belo Horizonte, MG, Brasil.

A survey on the efficacy of 6 vaccines commercialized in Brazil against Clostridios is revealed a very weak or no protection in neutralization tests performed in mice (Azevedo, E.O., Lobato, F.C.F., Abreu, V.L.V, 1998, Arq. Bras.Med. Vet., 1998, v. 50, n.3, p.239-242). Most vaccines are toxoids from concentrated culture medium where the toxin is present in low amounts. The use of purified toxoid would allow for better formulation and immunogenicity of the vaccine. The epsilon prototoxin was purified by ion-exchange chromatography on DEAE-Sepharose and a single band was observed by SDS-PAGE and silver staining. The incorporation of cold iodine (Heneine, L.G.D., and Heneine, I. 2001, Toxin Reviews, v.20, n.3/4, p. 209-228) to the prototoxin resulted in the detoxification of the activated toxin by trypsin, showing no lethality when injected intravenously in mice. Serum from sheep immunized with the iodinated toxoid had high anti-epsilon titres when tested by cELISA. The purified prototoxin shows a high level of denaturation (Worthington et al, 1973. J. Vet. Res. v.40 p.143-152), a lot kept at –20 °C was totally denatured after 6 months, when evaluated by its protein content, using the coefficient exctintion method and another lot kept at –80 °C decreased its protein content by 50%. The iodinated toxoid had increased stability when kept at –20 °C and when kept at –80°C there was no decrease protein content measured by the exctintion coefficient and by the appearance of denaturation bands on SDS-PAGE. The iodinated toxoid could be tested as a vaccine antigen.

Acknowledgements: CNPq, FAPEMIG.

CORRESPONDENCE TO:

HENEINE, L.G.D., Immunology Laboratory, Fundação Ezequiel Dias, Rua Conde Pereira Carneiro, 80, Gameleira, Belo Horizonte, MG, Brasil, Email: heneinel@funed.mg.gov.br