Part 3, Dr. Robert Harrison: Improving the Anti-venom

This part of the talk is fully dedicated to possible ways to improve the quality and safety of snake anti-venoms (AV). As we knew from last video, AV is a product about hundred years old, that it is manufactured following approximately the same procedures. Only 10 percent of the AV is IgG effective. The rest are only  proteins that contribute to the multiple adverse effects. Let us see what they did increase specificity, efficacy and safety of snake AV.
Very interesting.

Part 3 starts at minute 18'

This is a 10 ml, most vials of AV are 10 ml, that's the dose for instance that we gave for EchiTab G, one of the few AV that you only require one vial to effect complete cure in over 75% of the patients and if you give two vials is a 100%. Is a really great AV but, take a look at this, actually, is really poor because only 10%, between 10 and 15% of IgG in a vial of AV is specific to venom proteins. So, that means 90% of your drug is completely redundant and all is doing is contributing to the level of adverse effects in your patients. Right? Now, when we take a look at the venoms proteins (two-dimension electrophoresis) its quite clear that many  AV proteins aren't not pathogenic so, of your 10 ml, only 0,5 ml is effective at neutralizing pathology. There is a huge redundancy in this system that we think can be tackled by science.

The other think that we need to know is that the efficacy of current AV is restricted to the snakes whose venom was used in its manufacture. So this AV made against saw scaled viper is very effective for saw scaled vipers but is absolutely useless for mambas or cobras. However, in continents like Africa, you have a pretty large number of snakes so we know that through out all sub-saharan Africa, if you wanted to make a good AV, applicable anywhere, you have to inject your horses with over the 20 venoms of 20 different types of snakes. That's massive. And this is the problem with that, the greater the need of a poly-specific, multi-country AV, the more venoms you need to give to immunize your horses.

The problem is the venoms are very complex in terms of their protein composition, and each of this proteins is going to give rise to a different and IgG specificity. If you give 3 venoms you probably are immunizing against fifty and a hundred different proteins, and you are asking the poor horse, its immune system, to make antibodies for each of those different proteins. What this does means also is less IgG for snake to treat your patients so to overcome that, what we do in the field is we give more vials of AV, so the greater the number of snake venoms used, the greater number of AV vials required to that treatment.

Unfortunately, the greater the the number of AV vials you give, the grater the risk, the more precipitates the drop in safety. Here a patient suffering from pruritus and abdominal colic.... And also of course, the more vials you give the greater the cost to the patient so, as I said before, it is not uncommon cost for a treatment to require three, five hundred, up to seven hundred dollars. We came across a  family in Kenya whose life was wipe out because they had to sell all their goods to paid for the AV to treat that children. So that family is now destitute as a result of the way AV are manufactured and the physical and safety implications that follow.

There is an urgent need therefore for research, to develop AV that are much better in terms of efficacy, safety and affordability and yet retain their poly-specific effectiveness, and that is what we a trying to do so this describes also the research and priorities to improve the efficacy, the cross-species clinical effectiveness, affordability and safety and also develop a treatment for the venom-induced tissue destruction.

DOSE-EFFICACY: We want to change a vial of AV so that every single IgG in there is effective at neutralizing a venom protein that causes pathology. This is the way we are going about it: this is the first study that we have done on this and is targeting all the saw scaled vipers in sub-saharan Africa.

Distribution of four Echis species in Africa and the Middle East. 

E. ocellatus – blue, E. pyramidum – red, E. coloratus – green,E. carinatus – purple.

This is their range, three main different species that we are targeting, what we are doing is following a genetic approach, first of all we isolate all the genes from the snake venom glands to create what is called the transcriptome, this is the description of all of the transcripts, the RNA transcripts in the venom gland of the saw scaled viper. We then are able to interrogate that data and make a decision, based on literature and on toxicity data, but is only the proteins encoded by these genes that we need to worry about. All these others are genes encoding non pathogenic proteins, so we focus on this slot here.

And what we have done then is take a look at all of the sequences, converted them with algorithms into ways that we could look at the predicted immunogenicity of domains within each group, so this is all of the genes coding for metalloproteinases, one of the most dangerous venom molecules around and we are looking now at them in terms of domains within them that are likely to be immunogenic and where they are conserved across different transcripts we are taking those out, we  identify them, because the point being is if we can make an antibody to this it would bind to that domain irrespective of which protein it comes from, it is present in, and we pull out, remove those sequences, stitch them all together and we have now that these different epitopes are then used to immunize a mouse. And we want to get the antibody back from this mice and what we have found is that once this mice generate the antibodies, its called the SVMP epitope string, we looked at if whether it was effective in a model, this is a horrible model but is the one we have to measure hemorrhage induced by venom proteins, here we mixed the venom and the test substances, this time PBS, together and then injected in the skin of a mice, 24 hours later you look at the extension of the dermal lesion, the hemorrhagic lesion you can see here is huge, and is very very florid. Take a look now at the same amount of venom but this time pre-incubated with IgG from this epitope string immunized  mice and you can see there is over an 80% reduction of the size of the hemorrhagic lesion but also, the floridity, the severity of the hemorrhage. So, a single immunogen has been created that neutralizes the most lethal effect of this viper.

Encouraged by that, we decided to continue this project but this time expanded from the snake venom metalloproteinases (SVMPs) and go to this other very dangerous groups of venom proteins (Disintegrins, C-type lectins, Serine-proteases, group II Phospholipase A2) 

and we did this now just in a slightly different variety, so this is now with recombinant proteins and what we wanted to find out is can we make these epitope strings that are producing IgG that are toxin specific? because this a central to our thesis. And we have here now are all of the venom proteins in all the saw scaled vipers of Sub-saharan Africa and we also are showing here some snakes that are not saw scaled vipers but they are vipers. That's how the protein content looks like. The next series of slides is which proteins are bound by the IgG that we raised in mice immunized with epitope strings and they should be specific, there should be clear differences between each panel and there are. This is epitope string one against metalloproteinases, you can see that there are some similarities but also there are very clear differences and very different also for the serine proteinases so, what we had been able to do here by carefully designing this immunogen, we generated IgGs that are specific in binding exactly what they are supposed to do. And we have done that for all of the major toxic groups as illustrated here and look at the specificity.

Now, next step was to combine all of these IgGs so we now would have a combination of anti-SVMP, anti-disintegrin, anti-PLA2, anti-serine proteinases, all combined and all used to decorate the same array of proteins and you can see here that virtually every protein in the venom of the saw scaled viper is bound by this pool of toxic specific antibodies and there is some binding to this proteins in these other vipers because there is some similarity from viper to viper.Look now what happens when you compare this array with EchiTab G, with the AV that we made for Nigeria and in terms of the Echis (scientific name for saw scaled viper family), actually they are very very similar. So, all this by immunizing mice on three occasions, so there is no way near the strength of the AV when you are immunizing a sheep on a monthly basis for over a year and a half, we are really encouraged by this. 

Unfortunately, because it is a mouse model we did not have enough IgG to test its efficacy in neutralizing the lethal effects of SB but we are really encouraged by this. We also know that what we have to do is expand this approach to all of the venom proteins to all the snakes of Africa and that is something that we are working up into grant application at the moment, so that we have continent wide efficacy, and we feel that is has to be a monoclonal antibody in nature and it should be humanized. If we can achieve that, we will increase the dose-efficacy by over 90% because every IgG in this vial will be toxin specific and that means that it will be highly effective and because it is humanized there will not be adverse effect response and if we can achieved all that I think we can get "pharma" interested in taking this on as a commercial product. That's critical because otherwise it might not be effect and if it is that good I think demand would be significantly high than it is at the moment. That's the rational behind it.

I will stop here. Min: 30:25. Next part will be on snakebite tissue necrosis. A very important point and how AV can be effective on improving the outcome of it.