One of the deadliest human-wildlife conflicts plays out in India every day: Snakebites kill around 58,000 people, and disable 140,000 people every year. Snakebite envenoming leads to around 100,000 worldwide deaths a year, which means India accounts for more than half of total deaths.
Treatment largely comprises administering antivenom. Despite the huge burden of death, treatment still relies on technologies that have hardly changed for more than a century. Traditional antivenoms are made by injecting snake venom into horses and collecting the mixture of antibodies the animals produce. India uses a polyvalent antivenom for all snake bites. However, the polyvalent antivenom is produced against the venoms of the Big Four snakes: the common krait, the spectacled cobra, the saw-scaled viper, and Russell’s viper. India doesn’t have antivenom for other deadly snake species. Researchers estimate 60 of the 300 species of snakes in India are medically important.
To tide over the downsides of the traditional antivenoms such as their efficacy for a limited number of species, their after-effects, researchers engineered antibodies that would neutralise venom produced by different species of cobra in India. Tested in mice, the cocktail proved successful. The study was published in the journal Science Translational Medicine.
This project brought together expertise in toxinology, antibody engineering, protein science, and translational biotechnology among researchers from Indian Institute of Science (IISc), Bengaluru, and Denmark, Thailand, and several other countries.
“The study shows how we developed a recombinant nanobody-based antivenom that protects mice against experimental envenoming by geographically diverse cobra and king cobra venoms from India, demonstrating the potential of rationally designed recombinant antivenoms as an alternative to traditional animal-derived products,” Kartik Sunagar tells Hot Rock. A corresponding author of the paper, Sunagar is an associate professor and principal investigator at the Evolutionary Venomics Lab at the IISc.
A recombinant antivenom is different from a conventional antivenom in that researchers choose specific antibodies that are known to neutralise important venom toxins and then produce them individually in the laboratory using biotechnology.
In the study, researchers combined five different nanobodies, each selected to recognise and neutralise particular types of toxins found in spectacled cobra, monocellate cobra, and king cobra venoms. A nanobody is a particularly small type of antibody originally derived from the antibodies naturally found in camelids such as camels, llamas and alpacas, Sunagar explains.
The five nanobodies together act against three major toxin groups: neurotoxins, which interfere with nerve signalling; cytotoxins, which damage cells and tissues; and phospholipases A2—a family of enzymes that act like molecular scissors, cutting cell membranes and snipping bonds between fat molecules, eventually leading to pain, swelling, and inflammation.
Sunagar says that the five nanobodies were deliberately selected and combined based on what we know about the composition and biological effects of these venoms, rather than creating an arbitrary mixture.
He explained how these small antibody fragments work: rather than facing off against the venom as a whole, the nanobodies collectively zero in on and neutralise the major groups of toxins responsible for the harmful effects of cobra and king cobra venoms, including toxins that cause paralysis, damage cells and tissues, and contribute to other effects of envenoming.
The researchers produced each of these nanobodies individually in the laboratory and combined them in defined proportions to create a single antivenom cocktail. They then tested whether the nanobodies could recognise venoms from spectacled cobras, monocled cobras, and both species of king cobras found in India, including snake populations from different geographical regions. They also examined whether the nanobodies could actually block the activity of the toxins, rather than simply bind to them, and assessed their stability and suitability for further therapeutic development.
Finally, they tested the cocktail in mice. Sunagar says in testing they applied the cocktail in two ways. At first, they mixed the venom and nanobodies together before administration to establish their ability to neutralise the venom. They then simulated a real-world scenario where there is a time lag between entry of the venom and administration of antivenom.
They tested treatment at different time points after envenoming and progressively reduced the amount of nanobody administered. Sunagar says this allowed them to determine not only whether the treatment could rescue animals after envenoming, but also how effective it remained when treatment was delayed and when much smaller quantities of nanobodies were used. The study showed that the treatment could rescue animals even when administered after envenoming, including with treatment delays of up to 20 minutes in the tested cobra model.
What this study provides is a proof of concept: effective antivenoms can be built from a well-designed, deliberately chosen group of nanobodies. Next step is testing it in humans.
The current cocktail of antivenom does not protect against all medically-important Indian snakes, particularly kraits and vipers, because their venoms contain important toxins that these five nanobodies were not designed to target.
However, Sunagar says, “The modular nature of the technology means that additional antibodies or other toxin inhibitors could potentially be incorporated to broaden its coverage. The work therefore provides a framework for developing more comprehensive recombinant antivenoms for India and other snakebite-endemic regions.”
A significant insight coming through the research is that antibodies that worked against African snake toxins could neutralise venoms from Indian snakes. As Anne Ljungars, another corresponding author of the paper, says, “It highlights how conserved many medically important toxins are despite the diversity of snake species. It (the work) shows that we can target shared medically relevant toxin families rather than designing antivenoms for each snake individually.”
