A new recombinant antivenom that targets multiple venomous snake species—including the African spitting cobra, shown here—could lead to both safer and more effective snake bite treatments.
From rubber tappers in Liberia to sugar cane farmers in South Africa, bites from venomous snakes are a constant worry.1 In sub-Saharan Africa—which is home to green and black mambas, spitting cobras, and vipers—more than 300,000 snake bites occur every year.2 Since many of these bites occur in remote areas, the reported greater than 7,000 deaths and 10,000 amputations annually are likely underestimates.
While antivenoms are available and effective at treating bites from many venomous snakes, the technology to make them has not advanced significantly since their initial invention in the 1890s. Creating antivenom requires immunizing horses with venom from a specific snake, purifying the antibodies from the horse plasma, and giving those antibodies to snake bite victims.
“They are definitely saving countless lives. However, I’ve seen people [in] firsthand experiences that they get into shock because a lot of a heterologous [substance] from another animal is injected into a human body, and the immune system is reacting,” said Shirin Ahmadi, a snake venom and antibody development researcher at the Technical University of Denmark. The presence of non-toxin targeting antibodies in horse plasma also lowers the overall dose of useful antibodies in the antivenom.
To overcome these flaws in traditional antivenoms, Ahmadi and her team at the Technical University of Denmark developed a new antivenom made up of just eight nanobodies that prevented venom-induced death in mice for 17 of the 18 medically relevant elapid species of snakes in sub-Saharan Africa.3 This new antivenom, which is a mixture of a small number of recombinant nanobodies that can be produced in vitro, could lead to both safer and more effective snake bite treatments.
“[A] few years ago if you had told me that you can have a few antibodies neutralizing venoms of snakes across the continent I would say no way,” said Kartik Sunagar, a venom researcher at the Indian Institute of Science, who was not involved in the study. “Research over the last few years has actually demonstrated that this is possible… [but] finding something that works across so many species is definitely challenging,” he added. “It’s very encouraging to see development of all these cocktails that provide continentwide coverage.”
Finding Nanobodies to Neutralize Venom Toxins
Snake venoms contain a complex mixture of proteins, some of which are neurotoxins that cause paralysis, cytotoxins that can result in significant tissue damage leading to amputation, and metalloproteinases that can lead to unstoppable bleeding. Multiple research groups, including Sunagar’s, have identified antibodies that can broadly neutralize a specific toxin family.4
To identify broad-spectrum antibodies that could neutralize multiple toxin families from different snake venoms, Ahmadi and her team started by immunizing one alpaca and one llama with venom from the 18 different medically relevant elapid snake species from sub-Saharan Africa. Llamas, alpacas, and camels produce small antibodies that have just one single domain, unlike human or horse antibodies which have four domains. These antibodies are called camelid heavy-chain-only antibodies or nanobodies. Because nanobodies are much smaller than horse antibodies in traditional venom, researchers hypothesize that they can reach tissues faster and prevent the venom from causing extensive tissue damage.
The researchers collected nanobodies from the llama and the alpaca and created a phage display library which they then mixed with venom fractions that contained the most relevant toxin protein families.
“The scale was really scary in the beginning,” said Ahmadi. “Would it be possible to address entire [toxin] families between all these different snakes?”
The team then expressed their candidate nanobodies in Escherichia coli and tested their ability to bind the toxin families in vitro. They found that many of them neutralized neurotoxins as well as tissue-damaging cytotoxins. To narrow down the best combination of nanobodies, the team mixed groups of nanobodies with different toxin families or whole venoms, injected the mixtures into mice, and evaluated mouse survival. In the end, they identified eight different nanobodies as their optimal antivenom.
Rescuing Mice from Snake Venom
Of the 18 elapid venoms tested, the only venom that the new recombinant nanobody-based antivenom could not neutralize was the venom from the eastern green mamba.
Wolfgang Wüster
To determine the efficacy of the eight-nanobody antivenom, the team pre-mixed each of the 18 elapid venoms with their cocktail and injected mice with the mixture intravenously. They saw that all of the mice survived except for the one exposed to the mix with the eastern green mamba (Dendroaspis angusticeps) venom. They then performed rescue experiments where they injected mice with venom from 11 different snakes before administering the new antivenom intravenously, which more closely mimics a natural envenoming. The team found that their nanobody cocktail prevented mice from dying due to six different venoms, delayed the effects of the venom or partially neutralized the venom’s effects in response to two venoms, delayed death for two venoms, and had no effect on D. angusticeps venom. In fact, when the team performed the same rescue experiment with currently available antivenom, Inoserp PAN-AFRICA, it only partially neutralized or delayed death due to the tested venoms.
Finally, Ahmadi and her team wanted to see how well their nanobody mix could protect against venom-induced tissue damage. They saw in both antivenom-venom pre-incubation experiments and in rescue experiments that their new antivenom significantly reduced or prevented tissue damage from three different venoms.
Ahmadi had been sitting in a restaurant with her sister and cousin when she got a message on her phone with a graph of the tissue data. “I remember that I jumped, and I screamed in the restaurant! And then I had to explain that these are the results that we were looking for, for a very long time,” she said.
While this eight-nanobody antivenom has shown very promising results in mice, Ahmadi and her colleagues would like to optimize it further by, for example, seeing if they can reduce the number of nanobodies in the mixture even more. Also, since a mouse and a human’s metabolism are quite different, the team also plans to assess the half-life and the kinetics of their antivenom in a larger animal model.
“[It] is amazing to be part of this because sometimes you cannot see actually what you are working towards, but here we have a very clear mission,” said Ahmadi. “I really hope to see that all these efforts are translating into a real impact for people that are in dire need of it.”
