Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D.

Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 µg rHCC and rHCD each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with recombinant formulations developed a protective immune response against the respective BoNTs as determined by a mouse neutralization bioassay with pooled sera. Purified recombinant antigens were capable of inducing 13 IU/mL antitoxin C and 21 IU/mL antitoxin D. Similarly, both the recombinant bacterins and the cell lysate formulations were capable of inducing 12 IU/mL antitoxin C and 20 IU/mL antitoxin D. These values are two times as high as compared to values induced by the commercial toxoid used as control, and two to ten times as high as the minimum amount required by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), respectively. Therefore, we used a practical, industry-friendly, and efficient vaccine production process that resulted in formulations capable of inducing protective immune response (neutralizing antitoxins) against botulism serotypes C and D.

Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system.

A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H(C)) of the BoNT/A heavy chain was cloned into the replicon vector (H(C)-replicon). Cotransfection of BHK cells in vitro with the H(C)-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, H(C) VEE replicon particles (H(C)-VRP). Cells infected with H(C)-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H(C)-VRP, mice were vaccinated with various doses of H(C)-VRP at different intervals. Mice inoculated subcutaneously with H(C)-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.

Epizootic botulism of cattle in Brazil.

The first diagnosis of botulism in cattle in Brazil and its epizootiology are reviewed. The high prevalence of the disease raised on phosphorus deficient pastures in Savanna regions has caused severe economic losses in the past. The temperature induced microcomplement fixation test (TIMCF) confirmed the clinical-pathological diagnosis in all of the 24 cases studied by this method. The most important reason why botulism has not been controlled satisfactorily in Brazil is the lack of an available effective vaccine (type C and D). Additional prophylactic measures are phosphorus supplementation and removal of carcasses from the pasture.

Characterization of proteins, insoluble at low temperature, produced by Clostridium botulinum type C and C. subterminale. Their antigenic relationship with a similar protein synthesized by C. botulinum type G.

The production of a protein insoluble at low temperature ("cryoprotein"), by cultures of Clostridium botulinum type G has been shown to be a metabolic characteristic also shared by C. botulinum type C and by C. subterminale. These new cryoproteins have been purified and some of their chemical and immunological properties studied. It was found that both proteins were chemically very similar among themselves and to the cryoprotein isolated from C. botulinum type G. All these proteins are formed by a single polypeptide chain of approximately Mr = 180,000, with closely related amino acid compositions, isoelectric points and do not contain either free cysteine or disulfide bridges. Homologous and heterologous radioimmunoassays established the existence of an antigenic similitude among the cryoproteins from C. botulinum type G and C. subterminale thus becoming the first purified antigens which relate both bacterial species. If the production of cryoproteins can be shown to be a generalized phenomenon within the genus Clostridium these substances would provide an important tool to examine immunological and genetical relatedness between strains in this bacterial group.