 Good evening everyone, today I'm going to briefly talk you through the steps that I'm taking in my PhD to rationally design a vaccine for the bacteria group A streptococcus based on this protein called the T-antigen. So immune system is this really complex, really clever machinery inside of you that protects you from disease causing microbes every day, every second of every day, but it actually has a memory of its own. So when you're exposed to something and you get sick, your immune system develops this really strong, really specific response to that organism that's causing your disease and it remembers that. So next time you see it again, it's primed, it's ready to go, you won't even know that you've got sick with that same organism. So what vaccination tries to do is it seeks to exploit this a little bit by giving your body an ideally harmless vaccine that protects you against a serious disease so that if you're ever exposed to that, you never even get sick. So here we've got a painting of the very first vaccination that we have recorded in 1796 and this is Edward Jenner injecting the son of his gardener with some pus taken from a cowpox mark from milkmaid. And there are several different types of vaccines and what he was doing is he was using a live vaccine. So he was putting a live infectious virus into this trial and luckily for everybody involved, he was actually protected from smallpox. We also use these killed or attenuated vaccines. So the microbe has been killed or weakened in some way to reduce that risk of causing disease. However, both of these methods still actually are using potentially infectious material, they're potentially allergic. These are very, very low risk but they do happen. So with the advent of molecular biology, what we've been able to do is focus down exactly what the immune system is seeing on these bacteria. Got an example here, we've got something on the bacteria that the immune system is targeting and that's what it uses to actually protect you. So rather than needing to put in a whole bacteria or a whole virus, we can just put in this single subunit and this is what I'm going to be talking about today. So the bacteria we're trying to protect you against, it's called Grupe striped cocus shown yellow up here and it causes this really, really broad range of disease, some of which we would have all had, they're quite mild, pharyngitis and butygo and then to these really quite rare and nasty looking diseases such as flesh eating bug disease, still has a mortality of around 70 to 80% even today. But what we're really concerned about here in New Zealand is the fact that we have the dubious honour of having the highest recorded rates of acute rheumatic fever in the world and this is almost solely focused in our Māori and Pacific Island children. And what happens with rheumatic fever is you get infected with Grupe striped cocus and several weeks later for some reason which we don't understand and some people, the immune system goes haywire and what it starts doing is it starts attacking the heart and up here you can see we've got a healthy heart. I've just pointed out a normal heart valve. This is what opens and closes to pump blood around your body. When the immune system attacks it, you essentially get scar tissue forming on there. It can't open, it can't close properly and a lot of these patients will die from heart failure before middle age. So what we're trying to use to protect these patients from this is a protein called the T-antigen. So here we have three bacteria in the black and they're surrounded by this fuzzy hair like substance and these are actually individual fibrils called pilae and just like our hair is made of a protein called keratin, these pilae are made of several proteins and what they have is that one end, they've got an anchor that holds them to the bacterial cell wall and at the other they have a protein that's sort of like a hand and this is what the bacteria uses to latch onto our cells when it attacks us. But in between is this long thin fibre made out of one protein, repeated tens or hundreds of times so the immune system sees this many, many times. And this is the structure of one of those solved in 2007 shown here. And biological structures are important to us, they show us atomic level detail of these things, it shows us what they actually are and this tells us about what they're doing and how they're doing it and in this case how the immune system is interacting with that because that's what's most important. So the first step in designing a vaccine is you need to know what you're actually fighting, what is out there and what does your vaccine have to protect you from. So a few years ago a postdoc in our lab sort of did a survey on all the types of group A streptococcus that were circulating in Auckland as well as overseas and this is a genetic tree summarizing that work and the key finding there is that group A strep actually has 18 different types, there are 18 different strains. What that tells us that our vaccine has to protect you against every one of those strains so a vaccine has to protect against 18 strains otherwise there's no point. But the second finding from this that's important is that T1, I've shown that ringed in purple that was that structure I showed on the last slide that's actually isolated genetically, it sits off here on a branch by itself whereas the majority of them actually fall in this yellow cluster so they're far more closely related to each other than these guys out here. So for my work I've been using protein ringed in red called T18 and that's a representative of this yellow cluster so that also lets us look at differences, broad differences between these. So once we know what's out there we need to know how the immune system actually sees what's out there. So what I've done is we've vaccinated some mice and a rabbit with protein called T18 shown there and where there is a red square that animal has a really strong immune response to that protein. So we've done a blood test essentially against these different strains and what you see is that there's a really strong response to T18, our vaccine, but there's also a really strong response to a couple of others say three and 13 and another 18. So what this tells us is that immune system sees these in clusters so we can give it one and trick it into thinking it's actually seen four. So what for our vaccine design what this means is we may not actually need 18 different components in our vaccine to protect against those 18 strains. If we can select a smaller number of key proteins then they can each protect against three or four so that simplifies what we need to do for our vaccine. So the third step is we need to know what our proteins that we're putting in actually look like. So we know what T1 looks like but that's quite separate. We don't know what any of these others look like. So to do this I'm using a method called X-ray crystallography and this involves crystallizing your proteins and I've got a photo here down a microscope of some of the actual crystals that I've made and we shoot this with a very high-powered X-ray beam and when this hits the crystal it diffracts and is detected on a detector sort of similar to old-fashioned film and the pattern that these spots give you can tells you gives you information about what's actually inside that crystal and with some complicated maths that a computer can do for us can actually give us that atomic structure. So this is what I've done for the three proteins here shown in the greens and the blues and these are all from that big yellow cluster. They're quite closely related whereas the purple is this structure solved in 2007 that's quite different. You don't need to know anything at all about looking at biological molecules you can quite clearly see that all of these are incredibly similar if not identical and this is made even more remarkable because of the very low sequence identity. So this is the number I've shown in arrows. So this is telling us about the similarity of the building blocks that have been used to make these. So it's like we've given the purple bacteria a handful of Lego blocks it's built this structure and then you've given the blue bacteria the same number of Lego blocks but only 38% are the same and yet it's made a structure that is almost identical. So this tells us that this shape is very very important to the bacteria it needs this shape for survival to keep that pillar sculling to keep that hand there to grasp onto ourselves which also tells us that this shape is not likely to change so it's not likely to try and escape our vaccine by changing the structure. So we know that they are actually very similar despite being genetically quite different so we want to know exactly where these similarities are and where the differences are. So what we've been looking at so far is cartoon images which have been the skeleton so it's like looking at me and only seeing bone and that's shown in the ribbon in this green protein but around it in the sort of the transparent is a transparent shell and that's the surface that's like looking at my skin so that's what the immune system is actually going to see from the outside as it attacks this protein. We've also got a I'm comparing this to another protein another T-antigen here and anywhere this is green it has the exact same building block as in this protein on the left anywhere that it's blue the building block is different. So this is showing us similarities and differences on the surface that the immune system is going to see. So when we expand this to cover that whole tree all the diversity of group A strip we can see that on all of these they're being compared to T18 down the bottom here we can see that on all of these pilli there are regions of blue there are regions of difference this is a genetic difference that separates them into the 18 strains that's what we expect to see but most importantly of all every single one of these pilli has patches of green and that means there's the same patch on this pilli is the same as on this pilli is the same as on this so if we can train there or convince the immune system to attack one of those green places then you can potentially potentially protect against a very very wide range of strains using a very very small number of these proteins. So how does this all fit together what have we done we've identified T-antigen diversity we know what's out there we know how the immune system sees what out there we've solved the structures we know what our vaccine is we know how they're similar we know how they're different what I'm currently doing is looking at where antibodies actually bind single antibodies binding at the atomic level and putting that into assays that are testing whether those antibodies actually kill anything whether they're actually protective thank you