Gamma irradiated protease from Echis pyramidum venom: A promising immunogen to improve viper bites treatment

Echis pyramidum (Epy) is a venomous snake belongs to Viperidae family; it causes fetal coagulopathy systemic effects and death. Searching for more effective and safe antivenom is mandatory for viper bites treatment. Proteases are the most lethal components in viper venom inducing hemorrhage, edema and coagulation problems. Thus, the study aims to evaluate the potency of the prepared antisera and their neutralizing properties against the biological activities induced by whole Epy venom individually. Echis pyramidum metalloprotease enzyme (60 kDa) was purified using size-exclusion followed by DEAE-ion exchange chromatography. The purified Epy metalloprotease enzyme (SVMP) was detoxified with 1.5 kGy gamma rays from cobalt⁶⁰ gamma cell and used for immunization. 1.5 kGy irradiated Epy metalloprotease (SVMPi) showed less lethal activity (LD₅₀) compared to the corresponding native immunogen. The prepared antisera boosted against whole Epy venom (WV), 1.5 kGy irradiated whole Epy venom (WVi), SVMP and SVMPi were tested for neutralization of lethality and biological activities induced by Epy venom. The antibodies elicited against WVi and SVMPi were 30,000 and 20,000 EU, respectively. The anti-SVMPi serum showed the highest neutralization of lethality (ED₅₀) compared to the other prepared antisera. In addition, it prolonged the clotting time from 49.0 ± 2.5 to 176.2 ± 1.4 s. Furthermore, it demonstrated a highly neutralizing activity against edema induction and hemorrhage of Epy venom by 66.8% and 94.3%, respectively compared with the other prepared antisera. These findings would encourage further studies for using gamma irradiated purified fraction(s) from different snake venoms as safe antigen(s) to produce more effective antivenoms.