Exploring the Utility of Recombinant 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 in order 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 that 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.

Keywords: antivenom; immunogen; neglected tropical diseases; polyclonal antibodies; recombinant expression; serine proteases; snake venom toxin; snakebite.

Figure 1

The protein profiles of the purified recombinant toxins and their activity on human fibrinogen. Degradation SDS-PAGE gel electrophoretic profiles (reduced conditions, 8% gel) displaying the fibrinogenolytic activity of the recombinant toxins (3 μg, 1 mg/mL) following their incubation with human fibrinogen (3.75 µg, 2.5 mg/mL) at 37 °C for 120 min: (A) ancrod, (B) batroxobin and (C) RVV-V. PM represents the molecular mass protein marker, and the α, β and γ chains of fibrinogen are highlighted by corresponding labels.

Figure 2

Time-course analysis of the immunological cross-reactivity of pooled sera to the recombinant toxins and crude venoms used as immunogens over 14 weeks of murine immunisation. (A) Responses of anti-ancrod pooled mice sera against ancrod; (B) anti-batroxobin sera against batroxobin; (C) anti-RVV-V sera against RVV-V; (D) anti-toxin mix sera against a 1:1:1 mixture of the three recombinant toxins; (E) anti-ancrod sera against C. rhodostoma venom; (F) anti-batroxobin sera against B. atrox venom; (G) anti-RVV-V sera against D. russelii venom; (H) antivenom mix sera (positive control) against a 1:1:1 mixture of the three crude venoms. Data is shown for sera collected at weeks 3, 6, 10 and 14 (end of the experiment) of the immunisation time course. Non-immunised mouse sera (“normal mice control”) was used as a negative control. All mice serum samples were standardised to 1:50, then diluted fivefold. Data points represent means of duplicate readings, and error bars represent the standard deviation (SD).

Figure 3

EPT-ELISA analyses of immunological binding between the experimental antivenoms and the recombinant toxins and crude venoms used as immunogens. Each resulting experimental antivenom (anti-ancrod, anti-batroxobin, anti-RVV-V, anti-toxin mix and antivenom mix) is coloured differently, and their binding to the various toxin and venom immunogens are displayed in different panels, alongside data obtained with the normal mouse control negative control. Data shown represents binding levels to: (A) ancrod, (B) batroxobin, (C) RVV-V, (D) a mixture of the three recombinant toxins, (E) C. rhodostoma venom, (F) B. atrox venom, (G) D. russelii venom, and (H) a mixture of the three venoms. Each antivenom sample was serially diluted fivefold in duplicate, with data points representing means of duplicate readings, and error bars representing standard deviations (SD).

Figure 4

Immunological recognition of the toxin and venom immunogens by the different experimental antivenoms. (A) Reduced 15% SDS-PAGE gel electrophoresis and Coomassie blue staining was used to visualise the toxin and venom immunogens (ancrod, batroxobin, RVV-V, a 1:1:1 mix of these three toxins, and a 1:1:1 mix of C. rhodostoma, B. atrox and D. russelii venoms). The same venom samples were transferred to nitrocellulose membranes for immunoblotting experiments and incubated with 1:5000 dilutions of primary antibodies (1 mg/mL) of each of the experimental antivenoms, specifically: (B) anti-ancrod, (C) anti-batroxobin, (D) anti-RVV-V, (E) anti-toxin mix, (F) antivenom mix (as positive control) and (G) normal mouse IgG (as negative control). PM indicates protein marker and note that different molecular mass markers were used for SDS-PAGE and immunoblotting experiments.

Figure 5

Experimental antivenoms directed against recombinant SVSP toxins inhibit their fibrinogenolytic activity. Degradation SDS-PAGE gel electrophoretic profiles are displayed following the incubation of various samples at 37 °C for 120 min. Panels show different data obtained with the different recombinant toxins: (A) Ancrod, (B) Batroxobin and (C) RVV-V. For each, the following layout was used: Lane 1, protein marker (PM); Lane 2, human fibrinogen (3.75 µg, 2.5 mg/mL); Lane 3, fibrinogen (3.75 µg, 2.5 mg/mL) + recombinant toxin (3 μg, 1 mg/mL; ancrod, batroxobin or RVV-V); Lane 4, fibrinogen (3.75 µg, 2.5 mg/mL) + recombinant toxin (3 μg, 1 mg/mL) + experimental antivenom (1.75 µg,1 mg/mL; anti-ancrod, anti-batroxobin or anti-RVV-V); Lane 5, recombinant toxin only (3 μg, 1 mg/mL); Lane 6, specific experimental antivenom only (1.75 μg, 1 mg/mL): H represents IgG heavy chain (≈50 kDa) and L IgG light chain (≈25 kDa).

Figure 6

Inhibition of coagulation disturbances defined by the prothrombin time (PT). The assay measured the combined effect of the clotting factors of the extrinsic and common coagulation pathways (in seconds) in the presence of the recombinant toxins/crude venoms, and their recovery effect by adding specific experimental antivenoms/specific commercial antivenoms, incubated with FFP. (A) Ancrod, (B) Batroxobin, (C) RVV-V, (D) the experimental/commercial antivenoms controls and the normal mouse control, (E) C. rhodostoma venom, (F) B. atrox venom and (G) D. russelii venom. For each toxin/venom, “homologous” antivenom combinations were used (e.g., for ancrod the anti-ancrod antivenom was used as the anti-toxin antivenom, and the Malayan pit viper antivenom was used as the commercial antivenom). Each experimental antivenom alone, each commercial antivenom alone and the normal mouse control alone were used as negative controls. Error bars represent the standard deviation (SD) of duplicate measurements. The resulting data were statistically analysed with one-way ANOVA and Dunnett’s multiple comparison test. Values found to be significantly different to the venom + normal mouse control are indicated by asterisks: * p ≤ 0.05, ** p ≤ 0.01.

Figure 7

Quantification of fibrinogen concentrations following co-incubation of recombinant toxins and crude venoms with the specific experimental and commercial antivenoms and human plasma. The assay uses an excess of thrombin to convert fibrinogen to fibrin in diluted citrated human fresh frozen plasma (FFP). The resulting fibrinogen concentration is shown for each recombinant toxin/crude venom against the specific experimental antivenoms incubated in FFP. Data shown represents the following immunogens: (A) Ancrod, (B) Batroxobin, (C) RVV-V, (D) the experimental/commercial antivenoms controls and the normal mouse control, (E) C. rhodostoma venom, (F) B. atrox venom and (G) D. russelii venom. For each toxin/venom, “homologous” antivenom combinations were used (e.g., for ancrod the anti-ancrod antivenom was used as the anti-toxin antivenom, and the Malayan pit viper antivenom was used as the commercial antivenom). Each experimental antivenom alone, each commercial antivenom alone and the normal mouse control alone were used as negative controls. Error bars represent the standard deviation (SD) of duplicate measurements. The resulting data were statistically analysed with one-way ANOVA and Dunnett’s multiple comparison test. Values found to be significantly different to the venom + normal mouse control are indicated by asterisks: * p ≤ 0.05, ** p ≤ 0.01.