Despite being the non-target species, accidental snake envenoming in humans has resulted in hundreds of thousands of deaths and disabilities worldwide. Snake envenoming affects between 4.5 to 5.4 million people globally, inflicting over 100,000 deaths and 400,000 disabilities, annually [4]. Tragically, India accounts for 58,000 snakebite deaths and three-times as many immutable morbidities, making it a major snakebite hotspot [5]. Snakebite disproportionately affects the impoverished rural populations that often lack essential health infrastructure. As most bite victims are the primary breadwinners of their families, snakebite devastates far greater numbers of lives and livelihoods than currently recognized. Although snakebites kill nearly as many people in India as HIV infections, they only receive a fraction of the research attention and medical investment devoted to HIV. Since snakebite primarily affects the poor, and young males are at the highest risk of getting bitten, it results in severe socioeconomic consequences in developing countries. These considerations have led to the enlisting of snake envenoming as a high priority ‘neglected tropical disease’ (NTD) by the World Health Organization (WHO) [4].

On the flip side, snake venoms have saved many more lives than they have taken. Millions of years of evolution has resulted in diverse snake venom toxins with remarkable target specificities, and this property is being extensively exploited for drug discovery. Many snake venom proteins have been engineered into highly specific and efficient lifesaving drugs. For instance, Captopril, an angiotensin-converting enzyme inhibitor used for the treatment of hypertension, is derived from the venom of the Brazilian pit viper, Bothrops jararaca, and has become the poster child for drug discovery from snake venoms. This exceptional drug has saved millions of lives globally since its introduction in the early 1980s. Many other snake venom-derived therapeutics for the treatment of various diseases, including multiple sclerosis, thrombosis, and cardiovascular diseases, are under various phases of clinical trials [6].

Antivenom is the mainstay treatment of snakebite, whose manufacturing protocols have remained essentially unchanged since their inception in the late 1800s: purification of immunoglobulins (IgG) from horses hyperimmunized with sublethal and subtoxic doses of snake venom (Fig. 1). The efficacy of conventional antivenom is restricted to the immunogenic potential of venoms used in its manufacture. Since venom is an adaptive trait that underpins various quotidian functions, it often exhibits dramatic inter-(between) and intra-specific (within species) variability. This variation may result in very distinct clinical outcomes and, thus, severely limits the cross-population/species antivenom efficacy – i.e., treatment of snakebites of one population/species using antivenom raised for another. However, for the commercial production of Indian antivenoms, venoms are exclusively sourced in Tamil Nadu 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). Moreover, India is abode to many other medically important snake species capable of inflicting fatalities and morbidities in their accidental human bite victims. Northeast India, for example, is devoid of the ‘big four’ snakes, but is dominated by other medically important snake species. Unfortunately, however, a single polyvalent antivenom manufactured for treating bites from the ‘big four’ snakes is marketed throughout the country, including in regions that lack these species. As Indian antivenoms fail to account for the inter- and intra-specific variability in venoms, they are preclinically shown to be less effective in mitigating bites from the pan-Indian populations of both ‘big four’ snakes and the ‘neglected many’, medically important snakes for which antivenoms are not manufactured [7,8].

The manufacturing process of Indian antivenoms involves a) sourcing of venoms from the ‘big four’ snakes in a couple of districts in Tamil Nadu, followed by b) the immunization of healthy equines with these venoms in sublethal and subtoxic doses; c) immunized equines are then bled and the plasma is separated from the blood. The processed blood without plasma is mixed with saline and often reintroduced into the immunized animal; d) The serum is first digested with pepsin to cleave off immunoglobulin heavy chains, resulting in divalent F(ab’)2 fragments, followed by the treatment with ammonium sulfate or caprylic acid to precipitate antibodies, and ultracentrifugation of the precipitate to obtain purified antibodies; e) The purified antivenom is formulated either in liquid or lyophilized form before being marketed for f) the treatment of snakebite victims. Fig. 1 The production of conventional Indian antivenoms.

Hyperimmunization of animals with crude ‘whole’ venoms, which often contain antigens and other impurities, is the major shortcoming of the conventional antiserum therapy, as it increases the amount of contaminant antibodies in the finished product. In fact, the proportion of therapeutically relevant antibodies in an antivenom vial may be lower than 10-15% of the content [9], necessitating the requirement of a considerably large number of vials for efficacious treatment. This, in turn, increases the cost of treatment to the point that it becomes unaffordable to many in low- and middle-income countries. Fortunately, Indian antivenoms are heavily subsidized by the government and are freely administered without charges in government hospitals. Infusion of substantial amounts of therapeutically redundant antivenom; however, leads to complications, including serum sickness and the fatal anaphylactic shock. It is, therefore, not surprising that nearly 80% of snakebite victims who were treated with the Indian antivenoms were found to exhibit multiple adverse effects [10]. This highlights the pressing need to increase the dose effectiveness of currently available commercial antivenoms in the country.

To date, hyperimmunized animal-derived antivenom remains the only available treatment for snakebites. The inefficacy of such antivenoms in neutralizing the toxic effects of distinct medically important species and their geographically disparate populations have been well-documented. In recent times, several innovative strategies are being explored to develop next-generation antivenoms with increased potency, paraspecificity, and cost-effectiveness. Some of these strategies have been briefly described below.

Phage display: It facilitates the identification of antibodies specific to toxins of interest. Phage display essentially involves biopanning of antibody phage display libraries against a particular antigen, in this case, venom proteins, followed by the amplification and enrichment of the antigen-specific library. Selected phages are used for infecting bacteria, which are then allowed to express toxin-specific antibody fragments. Specific antibodies against various toxin types can also be combined to form a biosynthetic oligoclonal antibody (BOA) cocktail, which exhibits less batch-to-batch variation and increased efficacy and safety profiles than the conventional antivenoms [23]. Phage display technology has been shown to be effective in characterizing antibodies against medically important snake venom toxins, including crotoxin, cobratoxin, and dendrotoxin [24].

Synthetic epitope strategy: Next-generation antivenoms containing toxin-specific antibodies could also be produced through novel immunization strategies, such as immunization with synthetic epitope-strings. Herein, strings of nucleotide sequences coding for specific regions of various toxins are cloned into expression vectors and injected into animals for eliciting toxin-specific antibody response [25].

Aptamers: The use of aptamers, oligonucleotides or oligopeptides that bind to target molecules with high specificity, have also been considered for the development of novel antivenom therapies [26]. This strategy can be advantageous over animal-derived antibodies in terms of production, affordability, and ethical considerations.

Mimotopes: Structurally mimicking regions of clinically important toxins known as ‘mimotopes’ can elicit immune responses and generate toxin-specific antibodies. Examples include mutalysin-II mimotopes that have been shown to neutralize hemorrhagic activity induced by Lachesis muta venom [27]. These mimotopes are usually identified from a phage display library and have high specificity and stability.

Acknowledgements: RR Senji Laxme and Suyog Khochare (Evolutionary Venomics Lab, IISc) for their inputs.

Contributors: All authors contributed equally to the manuscript.

Funding: KS: Department of Science and Technology (DST) INSPIRE Faculty Award, DST-FIST, DBT-IISc Partnership Program, and the DBT/Wellcome Trust India Alliance Fellowship.

Competing interest: None stated.

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