A comprehensive preclinical assessment of Viekvin antivenom: Evaluation of neutralization efficacy and cross-reactivity toward European snake venoms.

In this study, the equine-derived F(ab')2-based antivenom Viekvin, produced against V. ammodytes venom (Institute of Virology, Vaccines and Sera "Torlak", Belgrade), was evaluated for its cross-reactivity and neutralization of enzymatic and toxic activities of venoms from Vipera ammodytes, Vipera berus, Vipera aspis, Macrovipera lebetina, and Montivipera xanthina. To characterize the antivenom's activity, various in vitro assays were implemented, including enzymatic activity measurements of snake venom metalloproteinases (SVMPs), serine proteases (SVSPs), phospholipase A2 (PLA2), and L-amino acid oxidase (LAAO), as well as erythrolysis, coagulopathy, and local tissue damage assays. Results demonstrated dose-dependent neutralization of venom-induced proteolytic, PLA2, and LAAO activities, with over 80% inhibition of SVMPs and SVSPs, near-complete neutralization of PLA2, and partial inhibition of LAAO. Hemolytic, edemagenic, hemorrhagic, and myotoxic effects of V. ammodytes venom were efficiently mitigated. This antivenom specifically recognizes immunogenic proteins of V. ammodytes and cross-reacts with other medically relevant Viperidae venoms, as confirmed by SDS-PAGE, Western bloting, and competitive ELISA. Potency testing demonstrated that antivenom exceeds European Pharmacopoeia standards for V. ammodytes, V. aspis, and V. berus, as well as providing partial protection against Macrovipera lebetina and Montivipera xanthina, supporting its broad therapeutic relevance and highlighting the importance of functional cross-reactivity assays in antivenom evaluation.