Molecular characterization, antigenicity and immunogenicity of anatoxic polymeric forms conferring protection against scorpion venoms
Résumé
Two polymeric forms of Buthus occitamus tunetanus (Bot) G-50 and Androctonus australis hector (Aah) G-50 were obtained by controlled polymerization with glutaraldehyde. Their mol. wts, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exclusion high-performance liquid chromatography using radiolabelled toxin, ranged from 14,000 to 90,000 and 90,000 to 100,000 for Aah G-50 and Bot G-50, respectively. Modification of about 20% of the lysine residues yielded completely detoxified Bot G-50 fractions which retained the antigenic cross-reactivity with the native G-50 fraction, venom and toxins. High levels of specific antibodies against venoms and toxins were generated by immunization with prepared polymeric forms because absorbance values higher than 3 units were regularly observed by enzyme-linked immunosorbent assay with immune sera dilutions of 1/10,000. In vitro protection experiments demonstrated that immune sera from mice, horse and monkeys efficiently neutralized either Aah or Bot venom. Depending on the adjuvant and animal model species used, the neutralizing titre estimated by subcutaneous injection in mice varied between 20 and 40 LD50/ml. The in vivo protection assays showed that immunized mice could resist the challenge by six times the LD50 amounts of the toxic fraction. This protection was found to be long-lived. It was concluded that G-50 polymeric forms are highly detoxified and immunogenic, and should be useful for the production of potent polyvalent antivenom against scorpion toxins. They could also be considered for further studies towards the development of vaccine candidates.