So what we see here is one polypeptide, that results in two different biologically relevant manifestations. This is the molecule subject to antibody surveillance and this is the molecule that’s functional for receptor binding.
So what does that mean to the immune response? Well, nothing good. Many can be clipped right off. In fact, in a lot of vaccination studies, these sites can be immunodominant. You can see that any antibody that binds to these mucin-like domain epitopes is going to be cut right off in the endosome leaving a perfectly functional receptor binding core that is now antibody free. So those kinds of antibodies don’t neutralize.
The essential conserved sites are not well exposed. So for example, all of these filoviruses share the same receptor, so that’s a conserved binding site, it’s an essential site for the molecule. You’d love to target that with an antibody. It’s completely or partially hidden under the glycan cap and the viral surface, so the antibody might not see it unless you found a way to engineer the antibody.
Because of this conundrum, we are left with a bit of a puzzle that neutralization and protection don’t always correlate for Ebola virus. So neutralization is your ability to inactivate the virus in vitro. Protection is your ability to save the animal in vivo. So for example, antibodies like this, this is the human kz52 from the survivor of the Kikwit, Zaire outbreak neutralizes brilliantly and doesn’t protect the primate. Antibodies like these, including two that bind the mucin like domains, and one that binds up with the glycan cap don’t neutralize but they do protect the primate. So this doesn’t make a lot of sense, leaving you wondering what works here.
We had this result years before and it really cooled everybody’s opinion on antibodies against Ebola virus, maybe it wasn’t even going to be possible to protect animals but then it turns out that you can. These are quite protective even if you wait long enough for hemorrhagic fever to develop. The difference might be that these are given in a cocktail as this was given alone. So does that mean we have to have a cocktail? It’s the replicative capacity or the length of the number of spike of Ebola virus such that we need to have multiple antibodies against multiple sites and if so, which ones do we put together? I mean two-thirds of this cocktail is mucin, does that mean that mucin works? Or is this one the champ that binds the top? We don’t know.
Now in the field we have about 200 different monoclonal antibodies identified against the virus. What do you put together in a cocktail. Well now I’m going divert a little bit from my theme about the proteins of the virus and then tell you how we can use the structure to get at that problem.
This is the website that the viral hemorrhagic fever immunotherapeutic consortium – you will be able to find this link through Scripps very soon. What this is that more than 20PIs in 7 different countries have gotten on the same page. We put almost all the antibodies known against these viruses together in one pool. We blinded them and then compare them side-by-side to see what is more effective. We’re going to try to see if we can correlate efficacy in-vitro and in animals and – to understand how to put together the right cocktail. Right now we have three from the army in a cocktail that neutralized and we have three from Canada in a cocktail that neutralized what if the most effective cocktail is one from japan and one from the army and one from Hamilton. We won’t know until we put them all together. And so it’s nice that everyone is on the same page in the same study.
Okay. So until we make that cocktail, let’s assume that viral infection will proceed. So the next step of viral infection — after the viral membrane is fused with the host endosome membrane, genetic material enters and the virus starts to replicate.
Now, something important happens here. Most people die from Ebola virus infection – 50% to 90%. Some people live. What is the difference? The difference seems to be that those people that survived the Ebola virus infection tend to generate an early and strong immune response against the virus and the viral titer starts to drop by around day 4. Those people that ultimately succumb to the Ebola virus infection are more likely to be characterized by a very poor immune response and their viral titers get quite high — 10 to the 9 to 10 to the 10 per mil at the time of death. So for this decision point to occur by around day 4, that means that the innate immune system is quite important in making this decision of survival or not survival.
So what is the viral factor at play in this innate immune decision point? One of them is a protein called VP35 — viral protein 35 Kilodaltons, that’s how it got its name. It’s a component of the nucleocaps in replication complex. That’s his day job. It also has another job – it’s interferon antagonist. And what it does in that function is to bind double-stranded RNA. Now you typically would only have double-stranded RNA in the context of a viral infection. And so it is a pathogenesis through the molecular pattern. Your innate immune system has sensors that go looking for double-stranded RNA and when they sense they mount to antiviral response, not having to know what the virus is, just the presence of a viral infection.
So how does one this work? This is a crystal structure of VP35 bound to double-stranded RNA. So the double-stranded RNA here is an 18-bp oligo in green. We have got four copies of VP35 bound to it. Now this half is identical to this half in the structure. So you can really only look this half if you want.
This is not the mode of ligand binding that you learned on your mother’s knee as a biochemist. So what you typically think of when you think of a protein binding of ligand is that it has one binding site. This laser pointer is a ligand. My hand is the protein, it binds in the palm and that is its binding site, perfectly shaped for it.
What we have here is the same protein binding in two different ways. Two copies bind the backbone, two copies cap the end. These are the identical proteins. You could pull off the end capping, want to roll it around and attach it by the backbone binding. They use different binding sites to do that. The end capping is a hydrophobic patch, and the backbone binding one uses a hydrophilic patch, this residue being central. So instead of the ligand binding in one site, you have two identical copies of the protein and one binds this way and one binds this way. It turns out that dimerization is essential, point mutations of block at dimer interface attenuate the Ebola virus.