 Good evening, ladies and gentlemen. My name is Sam Blanchett, and I've just come to the end of the first year of my PhD up at the Faculty of Medical and Health Sciences. My supervisors have been Associate Professor Thomas Profft and Dr Jason Lone, and tonight is a fantastic opportunity to share what I've been doing throughout the year, which has been working on my project, Vaccines Delivered on Bacterial here. So this probably raises a few more questions, and these are questions that I hope to answer over the next ten minutes. So first I thought I'd just start off by talking about vaccines. So there are many diseases out there where there is no vaccine available, whether it be a major exotic disease such as Ebola or malaria, or something a lot closer to home like a sore throat or a skin infection, something we've probably all had. There is no vaccine available for any of these diseases. So vaccines are needed in the community. They are the most cost effective, efficient way of treating diseases. They're so effective that they're actually able to eradicate diseases. You've probably never heard of anybody with smallpox. It's been wiped from the face of the earth with a successful vaccination program. In terms of medical and economic outcomes, it's better to prevent infections than to treat them. I know I'd rather have a few injections and spend days sick and off work. And with antibiotic resistance, it's a lot harder to treat infections, so prevention is becoming more and more our only option. So over the last few centuries, vaccines have developed quite significantly. This first image up here is Edward Jenner administering the first vaccine of live smallpox to an unsuspecting child. Good luck getting the ethics for that in today's world. So over time, vaccines have developed. We've realized injecting live pathogenic bacteria into somebody is not such a good idea. So vaccines these days tend to have bacteria or microbes that are significantly weakened that aren't healthy. We've also come to learn that we don't actually need the whole microbe to elicit a successful immune response. That's why vaccines such as the hepatitis B vaccine, sorry, use proteins found on the surface rather than the entire bacteria. Now we don't even need the whole protein. We can just use a small section of the protein and that's called a peptide. And peptide vaccines are the cutting edge of vaccine development today. Now like everything, they have their advantages and their disadvantages. Some of their advantages include the fact that there's no infectious material, so there's no risk of getting sick from this vaccine. And you're able to specifically target an immune response with this small peptide. Additionally, there's also no risk of an allergic reaction like there is with some conventional vaccines. Now of course there are disadvantages and some of these include peptides don't do anything. The immune system doesn't respond to them. They're unstable in the body, the body just chucks them in the rubbish. And to get them to elicit an immune response, you need to use potentially toxic adjuvants and these can require expensive chemical coupling. So that's where we're at with vaccine development in this day and age. So moving towards bacteria. Are bacteria actually hairy? Well not quite. They have these long hairlike projections across the surface of the cell wall. And these are called pilli. Now a pilli is one singular protein repeated over and over and over again. So in these long ones what we have is thousands of copies of this one protein. In these shorter ones there might only be a few hundred copies. But the take home message is that it's just one protein. So are all bacteria bad? Do they all cause disease? Well no. Each person in here has 10 times more bacterial cells in their body than their own cells. So by sheer number we're all more bacteria than we are us. Now this isn't a bad thing. Some of the bacteria are our friends, such as that found in Lactococcus, sorry such as that found in yogurt, Lactococcus Lactus. Now of course we can't be friends with everybody and there are bacteria out there that will do us harm. Such as those that cause infections like sore throats and in this case Streptococcus pyogenes. So the concept with PillVax, Pill is vaccine is to take these hairlike projections and put the genes responsible into the friendly bacteria. So what we end up with is this friendly bacteria, this Lactococcus Lactus, that expresses these hairlike projections from Grupe Streptococcus on its cell surface. Now the immune system sees these hairlike projections and recognizes the bacteria as unfriendly and thinks you want to do me harm so I'm going to destroy you. Now the good thing about PillVax is that the bacteria wouldn't cause us disease anyway so there's no risk of infection but the immune system thinks there might be. And if we include a peptide into this hairlike structure inside that one protein what we get is thousands of copies of this one protein expressed on the pillars. And the immune system will catch that peptide in the crossfire and will get an immune response to that peptide. So PillVax, if we go back to the advantages and disadvantages of peptide vaccines and put it into a PillVax concept, PillVax removes a lot of the disadvantages that we find in peptide vaccines. Because the peptide is contained within this highly immunogenic structure we get an immune response. This structure also protects the peptide from degradation so it's protected from the body and because the bacteria stimulates the immune system it removes the need for expensive chemical coupling and removes the need for potentially toxic adjuvants. We also get a bunch of bonus advantages. Bacteria are very cheap and very easy to grow. Some people don't like vaccines because they have a fair of needles. So we can administer this orally and we're also able to freeze dry this vaccine which makes logistics and distribution a lot easier. A lot of vaccines are temperature sensitive so being able to ship essentially a powder to places such as Africa where refrigeration supply lines are difficult and expensive makes this highly applicable and a lot cheaper than traditional vaccines. So when we take a gene from one bacteria and modified a little bit and put it into another we have to make sure that that new bacteria is actually expressing that gene and the way we do that is by carrying out a Western blot. So we extract the cell surface proteins from the bacteria and run it on a gel. This gel separates the proteins by size so at the top of the gel we have large proteins and at the bottom we have smaller proteins that have easily migrated through the gel. We then search for our protein of interest with an antibody and that antibody when we put it into a machine gives us a signal that comes up as a black line. So in striptococcus pyogenes the species where we're getting these pilli from what we see is this laddering pattern and if you remember back to this image where there are very different lengths of pilli that's what we'd expect. So because there are some large ones they're at the top of the gel and because there's some small ones they're at the bottom and there's a lot of intermediate sizing so there is this ladder. When we put this into lacticoccus we see the same thing so that means that this bacteria is expressing the gene successfully. This third line is just a control it's lacticoccus that hasn't been modified and you don't see any laddering as you would expect. So we know that Pilvax works from this pilot study that was carried out in our lab so on the y-axis what we have is just the level of antibody response and on the x-axis is the experiments that we carried out. So the first look sorry the first one we don't see any antibody response and this is where we've just injected the mice with a test peptide this test peptide is called OVA and we don't see a response and if you think back to the disadvantages of peptides this is expected because on their own they don't do anything. So with our Pilvax construct that we see in the blue that contains the test peptide we see quite a significant antibody response. It's comparable to this green one which is OVA that has been immunized with the traditional adjuvant cholera toxin B. Now this was just to test how effective Pilvax was compared to a more traditional conventional vaccination and we get a similar response but only when there's 50 000 nanograms of OVA compared to five nanograms in the Pilvax construct. So this means there is 20 000 times less peptide in the Pilvax construct which is quite amazing to elicit such a response. So now that we know that it works what we want to test for is how versatile this is what diseases we can use to vaccinate and one of the ones we've chosen is tuberculosis. Now there is a vaccine available for tuberculosis you might have heard of it some of you might have even had it the BCG vaccine but it's largely ineffective. This bacteria has been described by the World Health Organization as a global health emergency. It kills more people than any other bacteria and it causes quite significant and nasty lung infections. So the way we're going to target it is by targeting this ESAT 6 protein. Now ESAT 6 is essential for bacterial virulence in this disease. If you remove this gene from the bacteria it can't cause infection. If you complement this gene into a strain that doesn't cause infection all of a sudden it can cause disease. So if we target this and remove its ability to have an effect we should be able to prevent tuberculosis infection. So this is the whole protein and the bit we're targeting is just that small circled bit at the top the peptide the small section of the protein and when we incorporate the gene for this into our construct we want to make sure that the gene we've modified and put into a new bacteria is expressing. So we carry out the western block and what we see is this positive control with the laddering pattern and we again see the laddering pattern in our construct. So that our future work will be to vaccinate mice with this pilvax construct and to test for antibodies and other immune responses and to determine whether or not this vaccine offers protection. So there's a lot of exciting work ahead. Finally I'd just like to acknowledge my lab group for all their help and support as well as the faculty and the university. So what the immune system is responding to is not the bacteria itself it's responding to the pillow. So the pillow comes from Streptococcus pyogenes and this is a human exclusive pathogen that the immune system has evolved with over many many years and so it's come to recognize this as a as a dangerous signal and that sets off the immune response. The bacteria itself the Lactococcus doesn't really do anything and if you eat yogurt you're not going to get a response to Lactococcus anyway. So countries that we might want this vaccine in first. So tuberculosis is quite a worldwide issue it's estimated that about two-thirds of the world's population actually is infected with tuberculosis in a country like New Zealand that's going to be a very low number but in some places like Afghanistan or sub-Saharan Africa that will be up around nineteen ninety-five percent. So sort of the third world countries is where it would be best suited for first in terms of maximum effect. Distribution and administration that's still something that's been worked on but there are various ways you could do it you could include it in an ice cream or yogurt or all sorts of other things and you can ship it to these places quite easily because you can freeze dry it and it's basically in a powdered form that could be added to a number of things so it is quite easy to administer. So this is quite a novel technology that's been developed and recently patented by our lab so it is the first sort of thing out there like this. The only other similar thing I can think of is something by June Scott's group overseas who what they've done is they add their peptide at the very tip of the pilus. So the pilus like I said is that one protein for the most part. There are a few other proteins that anchor it to the cell wall and one at the top that helps an adhesion for pathogenesis and so what one group has done is to put their peptides at the top of the pilus but this isn't as effective because you want to get one copy per pilus whereas with our one you have one copy per protein in the backbone and because the backbone can have thousands of copies in one pilus or hundreds you get lots and lots and lots of copies of this peptide so there's lots of exposure and amplification of it.