Beyond the 'big four': Venom profiling of the medically important yet neglected Indian snakes reveals disturbing antivenom deficiencies

Abstract

Background: Snakebite in India causes the highest annual rates of death (46,000) and disability (140,000) than any other country. Antivenom is the mainstay treatment of snakebite, whose manufacturing protocols, in essence, have remained unchanged for over a century. In India, a polyvalent antivenom is produced for the treatment of envenomations from the so called 'big four' snakes: the spectacled cobra (Naja naja), common krait (Bungarus caeruleus), Russell's viper (Daboia russelii), and saw-scaled viper (Echis carinatus). In addition to the 'big four', India is abode to many other species of venomous snakes that have the potential to inflict severe clinical or, even, lethal envenomations in their human bite victims. Unfortunately, specific antivenoms are not produced against these species and, instead, the 'big four' antivenom is routinely used for the treatment.

Methods: We characterized the venom compositions, biochemical and pharmacological activities and toxicity profiles (mouse model) of the major neglected yet medically important Indian snakes (E. c. sochureki, B. sindanus, B. fasciatus, and two populations of N. kaouthia) and their closest 'big four' congeners. By performing WHO recommended in vitro and in vivo preclinical assays, we evaluated the efficiencies of the commercially marketed Indian antivenoms in recognizing venoms and neutralizing envenomations by these neglected species.

Findings: As a consequence of dissimilar ecologies and diet, the medically important snakes investigated exhibited dramatic inter- and intraspecific differences in their venom profiles. Currently marketed antivenoms were found to exhibit poor dose efficacy and venom recognition potential against the 'neglected many'. Premium Serums antivenom failed to neutralise bites from many of the neglected species and one of the 'big four' snakes (North Indian population of B. caeruleus).

Conclusions: This study unravels disturbing deficiencies in dose efficacy and neutralisation capabilities of the currently marketed Indian antivenoms, and emphasises the pressing need to develop region-specific snakebite therapy for the 'neglected many'.

Fig 1. Medically important Indian snakes and their venom profiles.

(A) and (B) are indicative of sampling locations and photographs of neglected snakes and their ‘big four’ counterparts, respectively. The map of India shown in panel (A) was prepared using QGIS 3.8 [20]. (C) venom samples, normalized for protein content (12–15 μg), were loaded onto 12.5% SDS-PAGE. Lane numbers indicate corresponding species (M: pre-stained protein ladder).

Fig 2. Comparative venom profiles of the medically important Indian snakes.

Venom compositions of Naja (A), Echis (B) and Bungarus (C) species are depicted as pie charts, and the relative composition of toxins are indicated in percentiles. A unique colour key for individual toxins is provided.

Fig 3. Distinct biochemical profiles of the neglected snakes and their ‘big four’ counterparts.

(A) Relative enzymatic activity of PLA₂; (B) Relative LAAO activity (absorbance units); (C) Specific hyaluronidase activity (TRU/mg/min); (D) Relative protease activity (%); (E) and (F) dose-dependent effect on activated partial thromboplastin time (aPTT) and prothrombin time (PT), respectively, where time (sec) is plotted against venom concentration (μg). All assays, with the exception of (E) and (F), were performed in triplicates and the standard deviation is indicated by error bars. A colour code is provided, which corresponds to the respective species of snake.

Fig 4. Binding efficacy of commercial polyvalent antivenoms against the venoms of medically important Indian snakes.

Binding efficacy determined using an end-point ELISA is depicted here. Optical density at 405 nm is plotted against various dilutions of antivenoms. The values are provided as mean absorbance of triplicates, and error bars represent standard deviation. The dotted lines represent antivenom titres, which were determined using purified IgG from unimmunized horses as negative control. Alphabets next to the dotted lines indicate the titre of the respective antivenom (P: Premium Serums & Vaccines Pvt. Ltd.; V: VINS Bioproducts Ltd.; B: Bharat Serums and Vaccines Ltd; and H: Haffkine Institute).

Fig 5. Identification of untargeted toxins via western blotting.

This figure depicts the results of western blotting experiments. Following the electrophoretic separation of snake venoms (25 μg) on a 4–20% gradient SDS-PAGE (A), bands were electroblotted onto nitrocellulose membranes, and incubated with 1:200 dilutions of various antivenoms: (B) Naïve IgG; (C) Premium Serums & Vaccines Pvt. Ltd.; (D) VINS Bioproducts Ltd.; (E) Bharat Serums; and (F) Haffkine Institute.

Fig 6. Toxicity profiles of snake venoms and neutralising potencies of commercial antivenoms.

(A) and (B) depict the median lethal doses of various medically important snakes and the neutralising potency of Premium Serums antivenom, respectively. Vertical solid and dotted lines in panel (B) indicate marketed neutralising potencies of antivenoms against N. naja (0.60 mg/mL), and B. caeruleus and E. carinatus (0.45 mg/mL), respectively.