Snakebite antivenoms step into the future

When two small children arrived at a local Tanzanian hospital with snakebites, Andreas Laustsen, then a university student, figured that they would be given a dose of antivenom, recover in the hospital for a few days, and then return home to their families.

“But what was done for them was that they were amputated,” said Laustsen, now an antivenom and toxicology researcher at the Technical University of Denmark. Doctors amputated one child at the elbow, and the other around the knee. “That made a quite strong impression on me.”

Snakes make their homes throughout the warm, tropical and sub-tropical regions of Africa, Asia, Latin America, and Oceania. Typically shy creatures, snakes are not interested in biting humans unless threatened or provoked. But in rural areas and in developing countries where many people work outside, accidental human-snake interactions are common.

Venomous snakes bite approximately 2.7 million people globally every year, resulting in approximately 138,000 deaths, with an even greater number of people experiencing permanent life-altering disabilities. Most snakebite victims are young, typically 10 to 40 years of age, and work in agricultural professions. Rubber tappers in Africa and southeast Asia, tea pickers in India and Sri Lanka, and rice paddy farmers in Myanmar are some of the people most affected (1).

“The burden of mortality from snakebite vast surpasses the burden of many infectious neglected tropical diseases such as leprosy, such as trachoma, such as filariasis,” said Abdulrazaq Habib, who is an infectious and tropical disease researcher at Bayero University Kano and leader of the Nigerian Snakebite Research Intervention Center. “It's not a high-profile disease,” he added. “The victims are voiceless.”

Antivenoms, which are made of animal-derived antibodies against toxins found in snake venom, are the only effective treatments against venomous snakebites, but even when snakebite victims manage to get their hands on them, antivenoms are expensive, have risky side effects, and are not always effective at neutralizing snake venom.

To find safer, more effective, and cheaper treatments for snakebites, researchers are developing new antibody-based and small molecule drug strategies. By focusing on expanding the number of snake species a specific antivenom can treat and by targeting both the cost and dangerous side-effects associated with current antivenoms, researchers hope to make snakebite treatments more accessible to the people who need them most.

The antivenom problem

Whether from the fangs of a red and yellow coral snake or the bite of the inky-tonged black mamba, snake venom is an extremely complex mixture. The most medically relevant snakes belong to the viper and elapid families. Venom from elapid snakes and some vipers contains neurotoxins, which block neuromuscular signaling. These toxins can eventually block signaling to the lung muscles, causing respiratory paralysis and death. 

Viper venom and some elapid venom, on the other hand, contain snake venom metalloproteinases (SVMPs), which weaken the walls of capillaries and blood vessels. These toxins can cause unstoppable bleeding and hemorrhage. SVMPs and toxins called phospholipases A2 (PLA2) can also cause tissue damage at the bite site, leading to muscle-weakening and tissue necrosis.

“Within those two groups [vipers and elapids], there's a lot of variation as well,” explained Nicholas Casewell, a venom researcher at Liverpool School of Tropical Medicine. “In Africa, a mamba and a cobra, they both cause this descending neuromuscular paralysis. They are both elapid snakes, but the actual toxins that cause that neuromuscular paralysis are completely different.”

Traditionally, pharmaceutical companies manufacture antivenoms by injecting small amounts of snake venom into a large mammal such as a horse, sheep, or camel over a period of months. Once the animal has mounted an immune response to the venom, the animal’s antibodies are collected and made into a vial of antivenom. But those can’t treat every potential snakebite. So far, there are only antivenoms available for about half of the known snake species.

“While current antivenoms are effective lifesaving treatments, they often, for several different scientific and biological and cultural reasons, don't get to the patients who need them the most,” said Robert Harrison, a snakebite antivenom researcher at the Liverpool School of Tropical Medicine. For example, a hospital must have the necessary antivenom to care for a bite from a specific snake species or group of potential snake species commonly found in the area.

Because antivenom is made up of animal antibodies, it’s possible that a person’s immune system can react to the foreign animal proteins in the antivenom, leading to anaphylactic shock or serum sickness. Due to this risk, physicians can only administer antivenoms in a hospital setting.

Antivenom is made up of about 10-15% of antibodies that will bind to and neutralize toxins in snake venom. This is because antivenom contains all of the antibodies that the horse or sheep ever produced in its life, including antibodies against any virus or bacteria it encountered. Therefore, doctors need to administer high volumes of antivenom to patients, increasing the risk of dangerous side effects.

“We can be talking about 10, 20 vials in some circumstances. Each vial consists of 10 milliliters,” said Harrison. “That's a problem because you get reactions to that antivenom in many, many people. And those reactions can rarely, thank goodness, be fatal, but they are clinically complex.”

For the farmers or their children who get bitten, leaving work to travel to the nearest hospital, which might take 10 or 20 hours, is often impossible. They may not be able to afford to leave their jobs for two to three weeks to recover in a hospital.

Antivenoms are also incredibly expensive to manufacture, which has led many pharmaceutical companies to halt production. Unlike other neglected topical disease treatments, snakebite antivenoms are rarely subsidized by governmental agencies, so the expense falls on the victims.

Recent advocacy by snakebite researchers led the World Health Organization to officially recognize venomous snakebites as a Neglected Tropical Disease (NTD) in 2017. In 2020, the Wellcome Trust awarded more than 80 million pounds to multiple research groups intent on developing new and improved treatments for snakebite. With the increased awareness surrounding the problem of venomous snakebite and the financial resources to do something about it, scientists are overturning every rock in search of new snakebite treatment solutions.

Making antivenoms a little more human

For John McCafferty, an antibody researcher at the University of Cambridge and founder of the antibody therapeutics company IONTAS, the key to making antivenoms safer starts in the horse.

“Although there are deficiencies with the equine antivenom antibodies in use, they nonetheless do save lives,” said McCafferty. “Somewhere within that horse is the answer to neutralizing snake venoms.”

Most of the horse antibodies target the most immunogenic proteins that the horse’s immune system has encountered. Because venom neurotoxins don’t necessarily stimulate a large immune response, horse antibodies against them will be less populous in the mix.

To identify horse antibodies that bind well to the venom toxins, McCafferty and his team first isolate monoclonal antibodies from the horse. Using a technique called phage display, of which McCafferty is a co-inventor, they create a library of bacteriophages that encode and express a single horse monoclonal antibody fragment. By exposing this library to venom neurotoxins, the researchers can identify the antibodies that bind the toxins the best.

McCafferty and his team then engineer those horse-derived antibodies to look more human by creating chimeric antibodies. The team swaps the horse antibody backbone with a human antibody backbone, but keeps the variable domains of the horse antibody, which specifically bind the venom toxin, the same.

“Essentially, three quarters of the antibody is now human,” said McCafferty. “The end result of our effort will be a cocktail of maybe six or twelve antibodies to very specifically the most toxic components of the particular venom presented as a human-like antibody.”

McCafferty admits that while this approach makes antivenoms safer, manufacturing these chimeric antibodies is expensive. But, he added, “nobody had the answer to human genome sequencing when they started the Human Genome Project, so one hopes that technology and improvements will make it feasible as time goes on.”

Laustsen, who often collaborates with McCafferty, also wants to improve antibody-based antivenoms by making them look more human, but he wants to leave all traces of the animal behind.

Our “recombinant antivenoms will consist of fully human antibodies,” said Laustsen. To find these human antibodies that bind snake venom, Laustsen uses a variety of selection methods. In the study described in a 2018 Nature Communications paper, he and his team used phage display to identify 25 human antibody fragments that bound to venom toxins from the deadly black mamba (2). When they converted the antibody fragments into human IgG antibodies, they found that a cocktail made from three of the human antibodies completely neutralized black mamba toxin in a mouse model.

Using a similar approach, Laustsen and his team recently reported their discovery of a monoclonal human antibody that can neutralize monocled cobra venom in a preprint (3). Starting with antibodies from humans who had never been bitten by a snake, they used phage display to screen for antibodies that could bind to the main toxin in cobra venom, α-cobratoxin, and they identified one that bound to it pretty well.

“It did show a little bit of neutralization capacity, but not enough,” Laustsen said. “After a while, we saw that it would let go, and the toxin would then jump off.”

To improve the antibody’s toxin binding ability, the team used a technique called chain shuffling. Instead of mutagenizing the antibody, which would make the antibody look less human, chain shuffling allows scientists to swap the light chains of different human antibodies to find the light chain pair that binds the toxin the best.

Laustsen likens chain shuffling to Dancing with the Stars: “There's a famous person and a not-so-famous person, and they get paired up. And the not-so-famous is the great dancer… We now know that ‘Jennifer’ is the best dancer, so we take her and then we just make a lot of copies. Then we let her take all the different partners to say okay, ‘what would actually be the best combination?’”

By using this approach, Laustsen’s team engineered a human monoclonal antibody that neutralized the monocled cobra venom in a mouse model.

“Some of the very strong critics, they still think you cannot make good enough antibodies unless you derive them from something that has been immunized,” Laustsen said, but this new preprint reported that, “yes, you can. If you use smart, synthetic biology techniques, then it's certainly possible.”

Moving forward, Laustsen and his team are engineering their human monoclonal antibodies to have greater cross-reactivity with toxins found in similar snake species. He is also excited about how broadly neutralizing antibodies for snake venom could have applications in other fields.

“Some of the stuff we're doing with broadly-neutralizing antibodies is immediately applicable for escape mutants from viruses or bacteria. They can be used to make drugs that don't, to the same extent, cause antimicrobial resistance in infectious disease areas,” he said. “I could imagine that you can make more complex precision therapies by having a mix between super-selective and broadly neutralizing antibodies, so that, I think, is exciting.”

Camel antibodies provide bite site treatment

While similar in stature to horses, camels have surprisingly small antibodies. Lacking the light chains that are typically found on antibodies in other species, including horses and humans, camelid single variable domain on a heavy chain (VHH) antibodies are very effective at binding and neutralizing their targets.

Their smaller size compared to full-size IgG antibodies means that they can diffuse quickly throughout the body, neutralizing snake venom toxins that cause tissue damage at the bite site. However, their diffusive characteristic means that they don’t stick around very long. When they enter the bloodstream, they will be quickly swept away from the wound they’re meant to treat.

To circumvent this, Harrison reasoned, why not administer camelid antibodies via the skin to treat tissue damage directly at the snake bite site? He was, however, concerned with how stable the camelid antibodies would be on an individual’s skin in the warm, tropical areas where snakebite occurs. In time, his experiments showed that even at high temperatures, VHH antibodies neutralized snake venom (4). 

“That suggested to me that with some clever further science down the road, you could actually formulate these VHH antibodies, which will be toxin specific, in ways that you could deliver them through the skin,” said Harrison.

Building on this work, Harrison and his team are immunizing camels in Kenya with only the specific toxins from African and Indian snakes that cause tissue necrosis at the bite site. The camels will create VHH antibodies against those specific venom toxins. Working with their partners at the International AIDS Vaccine Initiative, Harrison’s team will isolate B cells from the camels that produce the venom-specific antibodies.

The researchers plan to identify the best B cells and clone those antibody-encoding genes into bacteria to scale up their VHH antibody production. While this research is still in progress, the data the team have collected so far look very strong, Harrison said.

“It's quite possible that by binding to and neutralizing the venoms when they're in the skin, many of those venom proteins will never enter into the blood circulation. So, there is the potential that not only will we negate the tissue necrotic effects of snake bite, but we might also prevent the lethality,” he said.

For a safe and fast cure, bring on the small molecules

The best new treatment in the fight against snakebite might not actually be an antibody-based therapy at all, but a small molecule one.

Similar to camelid antibodies, small molecule drugs have the advantage of working quickly in the body and being relatively inexpensive to produce. With their quick mechanisms of action, small molecule-based antivenoms may be good solutions for treating people who live in rural areas soon after they get bitten.

“One of our strongest rationales for exploring drugs was the potential utility to be given as oral therapeutics,” said Casewell. “It might be that they're not sufficiently efficacious to solely treat the bite by themselves… but if it buys the patient time and just mitigates some of that acute pathology, we should still get a real measurable impact in terms of patient outcomes.”

Unlike antibodies, which target and neutralize a specific protein, small molecules can take out entire toxin families. This would potentially allow for a much broader neutralization of related snake venoms.

“It doesn't do anything to tackle the other toxin families. It's only going to target one, but if we start to add these drugs together, then we might be able to actually produce something that is actually generic and consists of very few molecules,” Casewell added.

Casewell and his team demonstrated this very possibility in a recent Nature Communications paper where they showed that by combining two small molecule toxin inhibitors, they could protect mice against venom from vipers in Central America, Asia, and Africa (5). One of the drugs, marimastat, is a matrix metalloproteinase inhibitor that was originally tested in clinical trials for cancer treatment. The other is varespladib, a phospholipase A2 inhibitor, which is undergoing testing for anti-inflammatory disorders.

“What's interesting is that some of the toxins in snake venom are also targets for other human diseases or conditions,” said Casewell. “So, we were really exploring those kinds of drugs where there might be sufficient overlap in the targets for us to see some inhibitory capability.”

By testing small molecules that have already passed Phase 1 or 2 clinical trials for other indications, Casewell and his team hope that any small molecule drugs that neutralize venom toxins effectively they find will be easier to bring to market and eventually to patients than new antibody-based antivenoms may be.

He and his team recently demonstrated that the phase 1 approved metal chelator, 2,3-dimercapto-1-propanesulfonic acid (DMPS) neutralizes a variety of viper venoms (6) and the venom from the Dispholidus typus snake species, a venomous member of the colubrid snake family (7). In fact, Casewell is now working with collaborators on a phase 1 clinical trial testing the safety of DMPS for snakebite in humans.

“Although the drug is licensed already, it's licensed for heavy metal poisoning, and heavy metal poisoning is quite different from snakebite in that it tends to be more chronic,” Casewell explained. “For snakebite, what we would be particularly keen to do is to dose it heavily in the first two days, and then probably nothing thereafter, so we want to understand how well tolerated it can be.” 

If there are no adverse reactions in the healthy Kenyan volunteers in the phase 1 study, Casewell and his team plan to move the drug into a small-scale phase 2 study to test its effectiveness at treating snakebite victims.

For Harrison, who often collaborates with Casewell, small molecule drugs have exciting potential as cheap, safe, effective, and easy-to-manufacture antivenoms.

“They're in tablet form, so they have none of the culturing problems that you have with [antibody] antivenoms, and if you have all of those together — safety, all of those things — then you can deliver that small molecule drug potentially to dispensaries, to pharmacies in the smallest towns in the world,” he said.

A snakebite free future

While new antibody therapies and small molecule drugs are on the horizon, snakebite victims are still in need of safe and effective therapeutics right now.

Because the pace of bringing drugs to market can be slow, especially for a tropical neglected disease like snakebite, José María Gutiérrez, an emeritus snakebite and antivenom researcher at Instituto Clodomiro Picado, expects that animal-derived antibodies will still be the main snakebite treatment for many years to come. Although many researchers like to say that animal-based antibody therapy is an old process, Gutiérrez pointed out that the technology has improved immensely over the years, cutting down the number of side effects snakebite patients experience.

“There are antivenoms of variable quality on the market, so some of them induce a high incidence of adverse reactions, whereas some of them induce very low incidence of adverse reactions,” Gutiérrez explained. “The goal here will be to improve the technology — especially in some developing countries — used for antivenom manufacture with technology transfer, with workshops, and qualification of the staff so that all these universal antivenom manufacturers improve the quality of the product.”

As new antivenom strategies are tested, scientists like Gutiérrez and Casewell are working to improve existing antibody treatments for snakebite. In an effort to increase antivenom’s marketability, and thus its accessibility to patients, Casewell and his team tested whether they could generate a broad-spectrum antivenom for snakes with venom that causes bleeding disturbances. The researchers mixed viper antivenoms in combinations of seven or twelve different antivenoms, and surprisingly, they found that the mixture of seven antivenoms protected against viper venom better than the mixture of twelve antivenoms (8). 

“We were looking at venoms that cause similar effects and ignoring geography in a way,” said Casewell. “The actual experimental antivenom we ended up with at the end of this process was by no means perfect, and in fact would need to be improved dramatically before it could be a product. But I think conceptually, there's something there that could be beneficial.”

Improving current antibody treatments and developing new treatment strategies is vital to reaching the World Health Organization’s goal of reducing the incidence of snakebite by 50% by 2030. But improved treatments alone will not be enough.

“You may have the best drugs in the world, but if they are not available and accessible for rural communities, then it wouldn't work,” Gutiérrez added.

To this end, the World Health Organization and snakebite researchers are working to help strengthen public health systems in countries where snakebite is endemic, to forge alliances with foundations and philanthropic organizations to provide financial support, and to engage with communities directly affected by snakebite.

Studies have shown that providing education around snakes and snakebite treatment to local communities and supplying transportation to hospital facilities reduces death due to snakebite in Nepal and India (9-10). Habib performed a similar study in rural communities in Nigeria. He and his team provided some communities with no intervention, education about snakebites, or education and an ambulance to shorten travel time to a hospital. He is working with his collaborators at the Liverpool School of Tropical Medicine to analyze the data, but he expects that the outcomes will be similar to prior studies demonstrating that community support improved snakebite outcomes.

In snakebite treatment, “there is a huge opportunity to make a real difference. We have very outdated therapies that save lives, but they have many, many limitations that can be circumvented,” Casewell said. “With the research we and others are doing, there is real opportunity to ultimately devise new therapeutics that are going to make a real tangible impact on the lives and livelihoods of people who suffer from tropical snakebite.”

References

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2. Laustsen, A.H. et al. In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies. Nat Commun  9, 3928 (2018).
3. Ledsgaard, L. et al. In vitro discovery and optimization of a human monoclonal antibody that neutralizes neurotoxicity and lethality of cobra snake venom. Preprint at: https://www.biorxiv.org/content/10.1101/2021.09.07.459075v1.full 
4. Cook, D.A.N. et al. Analysis of camelid IgG for antivenom development: Immunoreactivity and preclinical neutralisation of venom-induced pathology by IgG subclasses, and the effect of heat treatment. Toxicon  56, 596-603 (2010).
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6. Albulescu, LO. et al. Preclinical validation of a repurposed metal chelator as an early-intervention therapeutic for hemotoxic snakebite. Sci Trans Med  12, eaay8314 (2020).
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8. Alomran, N. et al. Pathology-specific experimental antivenoms for haemotoxic snakebite: The impact of immunogen diversity on the in vitro cross-reactivity and in vivo neutralisation of geographically diverse snake venoms. PLoS Negl Trop Dis  15, e0009659 (2021).
9. Sharma, S.K. et al. Effectiveness of Rapid Transport of Victims and Community Health Education on Snake Bite Fatalities in Rural Nepal. Am J Trop Med Hyg  89, 145-150 (2013).
10. Samuel, S.P. et al. Venomous snakebites: Rapid action saves lives—A multifaceted community education programme increases awareness about snakes and snakebites among the rural population of Tamil Nadu, India. PLoS Negl Trop Dis  14, e0008911 (2020).