Immune Response Against Bothropstoxin-I Irradiated With 60 Co Gamma Rays

Ionizing radiation has been successfully employed to modify the immunological properties of biomolecules. Very promising results were obtained when crude animal venoms, as well as isolated toxins, were treated with gamma rays, yielding toxoids with good immunogenicity. Ionizing radiation has proven to be a powerful tool to attenuate snake venoms toxicity without affecting and even increasing their immunogenic properties. However, little is known about the modifications that irradiated molecules undergo and even less about the immunological response that such antigens elicit. In the present work, we investigated the immunological behavior of bothropstoxin-I, a K49 phospholipase, before and after irradiation. Structural modifications of the toxin were investigated by SDS-PAGE and mass spectrometry. Aiming to compare the toxicity between native and irradiated forms of the toxin, an in vitro cytotoxicity assay, using CHO cells, was performed. Isogenic mice were immunized with either the native or the irradiated toxin. The circulating antibodies were isotyped and titrated by ELISA. According to our data, irradiation promoted structural modifications in the toxin, characterized by higher molecular weight forms of the protein (aggregates and oligomers). When analyzed by mass spectrometry, the irradiated bothropstoxin appeared in several oxidized forms. The citotoxicity assay showed that the modified toxin was 5 folds less toxic than its native counterpart. Irradiated toxins were immunogenic and the antibodies elicited by them were able to recognize the native toxin in ELISA. These results indicate that irradiation of toxic proteins can promote significant modifications in their structures, but still retain many of the original immunological properties.