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Snake envenomation results in a range of clinical sequelae, and widely used animal-based conventional antivenoms exhibit several limitations including the adverse immunological effects in human snake bite victims. Therefore, human monoclonal anti-snake venom antibodies or fragments can be an alternate therapy for overcoming the existing limitations. We developed venom-neutralizing humanized scFv antibodies and analyzed biochemical mechanisms associated with the inhibition of toxicity. Tomlinson I and J human scFv antibody libraries were screened against Naja naja and Echis carinatus venoms, and seven unique scFv antibodies were obtained. Further, specific toxins of snake venom interacting with each of these scFvs were identified, and phospholipase A2 (PLA2) was found to be prominently captured by the phage-anchored scFv antibodies. Our study indicated PLA2 to be one of the abundant toxins in Naja naja and Echis carinatus venom samples. The scFvs binding to PLA2 were used to perform in vivo survival assay using the mouse model and in vitro toxin inhibition assays. scFv N194, which binds to acidic PLA2, protected 50% of mice treated with Naja naja venom. Significant prolongation of survival time and 16% survival were observed in Echis carinatus venom-challenged mice treated with scFv E113 and scFv E10, respectively. However, a combination comprised of an equal amount of two scFvs, E113 and E10, both interacting with basic PLA2, exhibited synergistically enhanced survival of 33% in Echis carinatus venom-challenged mice. No such synergistically enhanced survival was observed in the case of combinatorial treatment with anti- Naja naja scFvs, N194, and N248. These scFvs demonstrated partial inhibition of venom-induced myotoxicity, and E113 also inhibited hemolysis by 50%, which corroborates the enhanced survival during combinatorial treatment in Echis carinatus venom-challenged mice. • Conventional antivenoms exhibit adverse effects in snakebite victims. • Seven human scFvs antibody fragments against two prominent Indian snake venoms were isolated. • Majority of antibodies were specific to phospholipase A2, which is an abundant toxin in both venoms. • These antibodies partially inhibited venom-induced myotoxicity, and one of them also exhibited partial hemolysis inhibition. • Antibodies developed against PLA2 exhibited partial in vivo venom neutralization. [ABSTRACT FROM AUTHOR]

Summary: The non‐specific lipid transfer proteins (nsLTPs) are multifunctional seed proteins engaged in several different physiological processes. The nsLTPs are stabilized by four disulfide bonds and exhibit a characteristic hydrophobic cavity, which is the primary lipid binding site. While these proteins are known to transfer lipids between membranes, the mechanism of lipid transfer has remained elusive. Four crystal structures of nsLTP from Solanum melongena, one in the apo‐state and three myristic acid bound states were determined. Among the three lipid bound states, two lipid molecules were bound on the nsLTP surface at different positions and one was inside the cavity. The lipid‐dependent conformational changes leading to opening of the cavity were revealed based on structural and spectroscopic data. The surface‐bound lipid represented a transient intermediate state and the lipid ultimately moved inside the cavity through the cavity gate as revealed by molecular dynamics simulations. Two critical residues in the loop regions played possible 'gating' role in the opening and closing of the cavity. Antifungal activity and membrane permeabilization effect of nsLTP against Fusarium oxysporum suggested that it could possibly involve in bleaching out the lipids. Collectively, these studies support a model of lipid transfer mechanism by nsLTP via intermediate states. Significance Statement: A model of lipid transfer mechanism of nsLTP from Solanum melongena has been proposed through intermediate crystal structures with lipid bound at different sites. [ABSTRACT FROM AUTHOR]