By Emmanuel Elebeke
Snake bite has recently been listed by World Health Organization (WHO) as one of the neglected diseases. It was removed from the list of life threatening diseases but now it is a problem. But on Wednesday, July 17, 2019, history was made when Africa Centre of Excellence in Phytomedicine and Development (ACEPRD), University of Jos, Plateau State announced the world first anti-snake vaccine. In this interview, the leader of the group that made the vaccine possible, Prof. John C. Aguiyi, and a member of his team tell the story of the anti-snake venom vaccine called COVIP-Plus. Aguiyi spoke first:•Prof. John C. Aguiyi
Tell us about COVIP-Plus?
The story of the anti-snake vaccine started in1994. It was an outcome of a research interest that had poor farmers at heart. As an NYSC member in 1989 at Lafia General Hospital, we saw several cases of snake bite including infestation in Shandam area of Plateau State, Kantungo then in Bauchi State and Taraba. I was thinking of how to curb this menace, and it occurred to me that the only way was to develop a drug management therapy. That I could not do until I joined the Faculty of Pharmaceutical Sciences at UNIJOS as a pharmacologist. The opportunity came in 1991 when I got a fellowship to do a post-doctoral degree in molecular biology at the International Centre for Genetic Engineering and Biotechnology Trieste, Italy. It was there that I found a journal of toxicology and, going through it, I saw several of publications in venoms and, behold, snake venoms featured prominently. As if what I was looking for before was brought up again, I took interest and collected several of the said venoms’ sequences with the intention of coming back to put those sequences together and produce an anti-snake venom vaccine. The vaccine idea was prompted by the fact that I was then working on HIV vaccine. When the programme was over and I came back to Nigeria, I could not follow up because there was no place to exercise the new experience.
Meanwhile I believe that whatever you intend to do will materialise one day, no matter how long it was delayed. So, in 1994 to be precise, one elderly woman was hawking seeds called Michiji, meaning worms or snake in Hausa. I bought some of the seeds from her and then, being the Head of Pharmacology, I went to search for the seed and it took me about six months to get this seed identified by another professor of botany in the Department of Plant Science. The moment he identified the seeds, I searched for it in my books, Trees and Evans. There, the description of the leaves for the management of snake bite was mentioned. I searched to see if any work had been done about snake bite management and there was nothing. I now had to start work. As a pharmacologist, I had to extract and test to see whether it relaxes tissues and, from there, to deduce what the functions are. I also tried to carry out the AD50, the later dose in every extract from plant source. The moment you establish the safety margin, you can also establish the dose from there. That was done and a dose was established.
The next step was to find out the reaction time, from what time does it have any effect, and we went further to discover that its effects manifests after 9hours. Then we never knew about its monological properties but we were just looking at it generally as a plant and its effect on venoms. Eventually, we discovered that after 9hours the extracts begin to neutralise venom effects.
How did you know that?
We injected the extract first, having established the dose and, from zero time, we were administering the venom to see at what time the neutralisation starts. So, we established that from 9hours most of the animals began to survive, and out of a group of eight, three died, three survived and that was increasing with time. We now established that, after 24 hours, when challenged, almost all the animals survived. That now made us to believe that this extract is effective against snake venom because the control with the later dose of snake venom, within two hours, all the animals will park up. The animals we pre-treated with the extracts, when challenged with the later dose of the same venoms, they all survived as against the control that will pack before two hours and it was a new and interesting finding. That was published and we have established that the claims were correct and that they were not empty claims. We tried to establish the neutralisation effect within 24 hours and found that some of the constituents were responsible for that.
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How did you clarify the structure?
Having established that, the next thing was to elucidate structure. It was ongoing when the idea of protein studies came up that snake venoms are mainly proteins. Here we have a seed that has 30% proteins, which is a large number. The next thing was to use vaccination method to inject the protein and, after two weeks, we gave a booster dose; then after 30 days, the anti-bodies were harvested in the form of conventional anti-bodies production method.
What animal was used?
Rabbit was used and Sera were inoculated into mice; after 15 days, a booster dose was administered and we then challenged. At the end, all the animals inoculated survived but the controlled all died. That gave us the impression that one of the major constituents responsible for the neutralization effect was the protein. We went further to study this protein and purified it. Initially we formulated it into tablets.
Does the vaccine apply to all snake venom?
Yes, it applies to all types of snakes including pythons. The time we initiated the studies, we had venoms of cobra, viper, puff ada and we applied all and they were all neutralised until there was nothing to establish again. The extract neutralised these venoms. The same principle was applied to the protocol. That was a good finding because the venoms I mentioned, some came from Pakistan, some from Brazil, these are international preparations and, of course, the venoms from Nigeria. We discovered that they were all neutralised. These are polyvalent proteins and so it does not matter the specie of the snake. Another good thing about this vaccine is that it does not only handle snakes, it handles bee stings, spider stings and scorpion and others.
We have a collaborator from Malaysia. When we were at the point of purification, the purification process was very difficult to establish and that took me to Malaysia and I spent one year in Malaysia trying to get a particular method for its purification, which we eventually got. By the time I came back, we tested some of the venoms in Malaysia, which has over 200 species of snakes and the most dangerous venoms, and the protein neutralised them. In fact, this story I am telling here would have been told in Malaysia but because of the way I saw them behaving. We are already looking into how to solve cancer problems and, with time, we have already established some molecules that have anti-cancer properties. After this, our next major focus will be on cancer. We have Stanford University in Manchester; University of Sienna in Italy, University of Malaya in Malaysia, etc. Here locally, we have Nnamdi Azikiwe University.
We have prepared a protocol, tested the vaccine in animals. We have done that in animals in the mouse, rabbit and sheep. Having done the testing in all these animals, now man, being much higher animal, we want to know the effect because man is going to be the end user. But the problem we have is getting clearance from NAFDAC because they are in charge of food and drug regulation and use. Once we get the clearance from them to test it on humans, we begin to talk of other things. It is getting to know the effect of this vaccine in man that is going to cost about N200m for the first phase and we have about four phases. The first one is to establish effectiveness and safety. Phase 2 is to see whether there is adverse effect and of course there are Phases 3 and 4. The most important phases are Phases 1 and 2. However, the major challenge is how to go into large scale production. We have formulated it into tablet form and tested its effectiveness.
We cannot be talking about adverse effect more so when we are talking about natural product. The locals swallow just two seeds and they are protected from any form of envenomation for one year. That also we were able to establish.
The conventional anti-snake vaccine we have in the market sells for N25, 000 – N35, 000 and here we are talking of a monovalent anti-serum; monovalent in the sense that it is prepared from inoculation of a known venom, whether that of viper or cobra, into the production horse. After 30 days, you can also harvest the anti-bodies and that is what we have in the market. It is because of the cost that we went into cloning. With cloning, within a short time, you can produce more than 50kgs with one roar but by formulation (tabulation) you will not produce as much as that. We looked at the purification process, the same process that was hindering the production of HIV vaccine. The bacteria cells cannot give a higher yield like the mammalian cells. In the bacteria, the protein expression comes with inclusion bodies; the protein of bacteria is fused into that of DNA. So, to separate them becomes difficult. The bacterial production proved to be very efficient.
Prof. Abraham Goni Dogo, Head of Laboratories on Prospects
This vaccine has prospect. This is something that has global impact. This is the first ever produced plant derived anti-snake venom in the world. Prof. Aguiyi should be given a Nobel Prize. We should push it even to the United Nations. The media should talk more about this, something that came from the bush and has global impact. Nigeria is emerging globally with this vaccine. That is to say, we still have researchers in this country that are resilient, patient to follow through to everything objectively to solve national and global problems. The prospect is that this will be accepted. A lot of research grants we have been expecting will flow into the country, and this, of course, will boost the economy and image of the country. It is something that can compete with advanced technology across the world. It can create jobs, bring in money. The polyvalent covers all the reptiles. It is something the world and Nigeria should embrace. The market is there in Nigeria, not to talk of Africa and the whole world.