Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms

Abstract

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

Exploring the utility of ssDNA aptamers directed against snake venom toxins as new therapeutics for tropical snakebite envenoming

“…To assess whether the binding exhibited by the highest affinity ssDNA aptamers were capable of inhibiting the functional activities known from SVSPs, we first used degradation SDS-PAGE to explore protection against fibrinogenolysis. Our findings revealed that, in line with the known functional activities of native ancrod and batroxobin and recent work (76) both recombinantly expressed toxins cleaved the α-chain of fibrinogen (Figure 4A and 4B). We then assessed the inhibitory capability of the two candidate ssDNA aptamers at preventing this functional activity.…”

“…The recombinant toxins used in this study, Ancrod from C. rhodostoma and Batroxobin from B. atrox , were previously expressed in HEK293 mammalian cell lines (76). In this study, we used these toxins to conjugate to NHS-activated Sepharose® 4 Fast Flow beads (Sigma-Aldrich, UK) to generate ssDNA aptamers as novel toxin inhibiting therapeutics.…”