 Good morning. My name is Alfredo Garzino-Demo. I have a faculty position at the University of Maryland Institute of Human Biology and Department of Microbiology and also the Department of Molecular Medicine of the University of Paldo in Italy. And I'm part of the Federation of European Microbiology Societies, FEMS, which is an association that promotes the advancement of science. And so this event is trying to bring novel advancement into the vaccinology of COVID and hopefully to inspire some young people. So FEMS also has some publications and this event is sponsored in particular by Microlife and a novel publication of FEMS. And I'm actually the editor-in-chief of another publication, which is another journal, which is Pathogens and Disease. Both of these journals Microlife and Pathogens and Disease welcome any submission in the field of virology and vaccinology, of course. Now we have a stellar lineup of speakers today and I'm very enthusiastic about introducing each of them. The first speaker today will be a doctor of chismachia or chisy corbett. She's a research fellow in the viral pathogenesis laboratory at the National Institute of Health and she works in the laboratory of Barney Graham, who is a virologist and a vaccinologist. Before that she was in the laboratory of Ralph Barrick at the University of North Carolina. And she got her undergraduate degree at the University of Maryland and in the laboratory of Barney Graham she started working on coronaviruses and now she has expanded the portfolio of viruses to a number of viruses besides corona includes influenza, dengue and other viruses. But where her work really took off is with what we call now the Moderna vaccine, which is the vaccine that actually I received. So thank you Dr. Corbett. I'm one of the vaccinees. And I love this story because of who Dr. Corbett is. And also the story of this vaccine is a vaccine that the other day I was in a webinar with another colleague that admitted that he was on the board of the company that put together the fighter vaccine and he said this will never work. And so the vaccine we are getting today is one of these history of sciences where something that shouldn't work works because people are persevering, you know. And from the title of the seminar I can tell that Dr. Corbett will tell us some of the history of this vaccine that I really want to hear and to be made public. So that is really her great contribution. She was one of the people that worked on the phase one trials, etc. And this was a vaccine that went from the viral sequence to the phase one to 66 days. The second speaker of today will be Dr. Florian Kramer. Florian Kramer got his training at the University of Natural Resources and Life Sciences in Vienna under the mentorship of Dr. Reiner Grapper, where he got his experience in expression of purification of glycoprotein. He then went for a postdoctoral work in the laboratory of Dr. Peter Palese at the ICAN Medical School in Mount Sinai, New York. And he did a lot of seminal work in the field of vaccinology of influenza. And when the SARS-CoV-2 pandemic happened, he was with people at the forefront of antibodies research. And today he will talk about some of that work. The third speaker is an incredibly accomplished vaccinologist, Professor Rino Rapuoli, who is chief scientist at the Head External Research and Development at GSK Vaccine in Siena, Italy. He's also a professor of vaccinology at Imperial College, an extraordinary professor of molecular biology at the University of Siena in Italy. He got his PhD in Biological Sciences at the University of Siena, Italy. I think a PhD in Biological Sciences is probably the only one thing that me and Dr. Professor Rapuoli have in common. And was a visiting scientist at Rockefeller University and Harvard Medical School. He's an elected member of the U.S. National Academy of Sciences, the American Academy of Art and Sciences, the European Molecular Biology Organization, and the Royal Society of London. So he has a distinguished career in vaccinology, introducing several key concepts including genetics detoxification, reverse vaccinology, and pan genome analysis. He also was one of the people that developed the first conjugate vaccine against meningococcus, developed the use of MF-59 as an adjuvant, and several other key contributions with the vaccinology. And he's also the founder of the GSK vaccine for global health. So these are the three fantastic speakers we have today, and I hope I did justice to their incredible achievement, but I am going to stop trying to take in time away from the presentation. And I will introduce Dr. Kizmekia Korbet. The title of the presentation is going to be SARS-CoV-2 mRNA vaccine development enabled by prototype pathogen preparedness. Thank you so much for that kind introduction, and thank you for the invitation to give this talk today. I wanted to spice it up just a little bit and include a part of the name of the webinar in my title. So I will be talking about rapid vaccine development in the time of COVID-19. And again, I am Kizmekia Korbet, and I am a senior research fellow in the laboratory of Dr. Barney Graham. I have the NIH in the Vaccine Research Center. So coronaviruses are a family of viruses for which we knew had pandemic potential. And the reason why we knew this is because the coronaviruses have spread unitically to humans before. This happened over a decade ago with the SARS epidemic that caused about 8,000 cases worldwide. It happened again around the time that I started at the Vaccine Research Center with MERS coronavirus that caused over 2,000 cases worldwide. So we had this historical reference for the pandemic threat that was coronaviruses, but largely ignored it. We even had science that directed us in that way, where Ralph Barrick's laboratory that as you said, I was at UNC during my graduate training, studied coronaviruses and how they might be poised for human emergence. He published a series of papers that specifically said that SARS-like coronaviruses, so that our viruses that are very much closely related to the SARS-1 virus might be poised for human emergence. So I like to think of the 2019 emergence of SARS-CoV-2 that has since turned into a global pandemic with 100 million cases worldwide to be the virus's way of saying, I told you so. So in knowing all that we knew about the pandemic threat of coronaviruses, there were several things by way of vaccine development that we needed. We knew that we needed a fast vaccine technology. So this is something that could be produced in vast quantities very quickly, something that was reliable. That is vaccine technology that appelled a reliable, manufacturability standard. And then lastly, and italicized, is universal. And I put this here because I actually am raised, so to speak, in the universal influenza field. And so understandably universal vaccines are vaccines that are preemptive and provide protection against future outbreaks. And while we're not there yet for coronaviruses, one of the things that I hope to translate in my talk today is that we at least had a universal solution. How do we get to that universal solution? The way that we did this is via what we call the prototype packaging approach to pandemic preparedness. Dr. Graham and I wrote a commentary on this approach in the Journal of Clinical Investigation. If you're interested, but really briefly, what this means is that you do enough research on a known virus within a viral family. You term this the prototype virus. In our case, we use MERS and we studied MERS for the last six years. You generate enough generalizable knowledge for that prototype virus that you're able to apply it to other viruses within that viral family. The second prong of this approach is that you would ideally develop the countermeasure, or in this case, the vaccine, through phase one clinical trials, which would help to speed development in the onset of a pandemic. We didn't necessarily do that with MERS, but again, we did have enough knowledge that we were able to, as I call it, plug and play in the onset of this pandemic. So the timeline for the rapid development of the SARS-CoV-2 mRNA vaccine. And I'm only talking about the specifics of the Moderna candidate now, although the timelines are fairly similar with the Pfizer candidate as are the technology that is used, including the spike protein. With that, we learned about what was then a respiratory virus outbreak in Wuhan, China in the latter parts of December of 2019. We then learned about the sequences of that virus that was then called 2019 in or novel coronavirus that were published on January 10th of 2020. So a little over a year ago, it was 66 days later that we entered to a phase one clinical trial. Actually, the anniversary of that is next week. And the really interesting thing about all of this is while the vaccine timeline from sequence to phase one clinical trial is record breaking. It is notable to say that there was extensive work not just from our laboratory or laboratories all over the globe that studied MERS coronavirus and other coronaviruses that really helped to fuel this vaccine development forward very quickly. So I want to talk a little bit about that extensive work or what is essentially the gray box of unknowns that many people had no idea was happening scientifically prior to the onset of the pandemic. So the viral the vaccine target is the coronavirus spike protein. It is this beautiful mushroom shaped protein that sits on the surface of the spike protein that you see on the left side of your screen. Its entire purpose is to engage with the human cell via what we call a functional cell receptor in the case of SARS-CoV-2. That is the ACE-2 receptor. Upon binding that receptor, it allows for the virus to then fuse with the host cell and then go into the host cell and replicate with that. Obviously, from the point the standpoint of vaccine development, you want to block that interaction in some way shape or form. And we generally think about that blocking of that reaction by way of antibody responses simply put. So we wanted to study that protein and we took knowledge that we gathered around many of the other proteins that have that same function for other viruses. So here are the panel, so to speak, of viral fusion proteins that look like and act like coronavirus spike proteins with the ability to allow the viruses to attach to the surface of cells. And so these fusion proteins have been studied extensively, particularly one that is, I consider to be the baby of my principal investigator, Dr. Barney Graham, who has studied respiratory syncytial virus for the entirety of his scientific career. And in studying the respiratory syncytial virus F protein or fusion protein, one thing became very clear. Is that if you stabilize these protein in their pre fusion confirmation and I will get into what that means, you can expose neutralization sensitive epitopes in its functional form. So what that means is that right here and sorry I don't have my some reason my pointer is not. Let's see if I can try to. Okay, so, as you can see here, there are two sides of the spectrum by way of the shapes of these proteins. The first is the post fusion confirmation. So this is after the protein has flipped into its post fusion confirmation is more elongated and relaxed. The first of the second is the pre fusion confirmation, where the protein is in a more squatted mushroom shaped form. The interesting thing that was really highlighted by proof of concept with the RSV field is that these red surfaces which are where the most potent neutralizing antibodies bind are very at top. They can show them to the immune system very readily with the pre fusion confirmation of the protein. However, they are occluded on the post fusion version of the protein. So obviously by way of vaccines, you would want to give this form of the protein to the body. So when we got when I got to the laboratory in 2014, the literature for coronavirus spike proteins was really a blank slate. We didn't know at any high definition what those proteins look like. And then following some some work that we did on human coronavirus HKU one, which is one of the coronaviruses that circulates endemically or seasonally around the time that flu circulates. We were able to solve the first pre fusion structure of a human coronavirus spike protein and we did this in a strong collaboration between Jason McClellan's laboratory and Andrew Ward's laboratory. At the same time, David Biesler's laboratory published a structure of the MHV coronavirus spike protein. And then the literature really just lit up with structures of coronavirus spike proteins detailing various things whether it be the glycan masking and the glycan shields and epitope masking, or how the proteins might flip from their pre to their post fusion confirmation. And then George Gow's group at the CDC in China published the cryo we am structures of SARS and coronavirus spike proteins were feeling some really cool features of those proteins that I don't have the time to go in today. But we also published the same, except the interesting thing about our publication is that we were able to utilize our knowledge. Based on all of the other spike protein structures, specifically our own HKU one structure to rationally design the pre fusion version of the MERS coronavirus spike protein and take that further to describe its immunogenicity. So that is how well does that pre fusion protein elicit immune responses in the pre clinical model. So the way that we did this is an exercise called structure guided stabilization. So you look at the sequence of the protein in this case is the spike protein and the one that we had defined structurally was the HKU one spike protein. You beg the question, where functionally does this protein flip into its post fusion confirmation. In the case of this particular protein, it flips by it right here at this hinge region, which is at the bottom of the protein and what we call the Sim or the S two region. And so plopping in some pro lanes in that hinge region, effectively stabilize that protein into its pre fusion confirmation. It allowed for the protein to be more homogeneous when you express it in cell culture. It allowed for higher expression levels of that protein. And as you can see here, very clearly, it's more mushroom and shape across the board, as opposed to in its wild type confirmation, this spike protein for MERS readily flipped into its post fusion confirmation. So we were able to not just do that for the MERS coronavirus with those same synonymous to pro mutations, but for SARS and HKU one, and it's interestingly enough for various other coronavirus spike proteins, including the potentially emerging MERS, which was called W IB one that Ralph barracks laboratory deemed to be poised for human emergence. So, taking this knowledge together, also in that paper defining that we were definitely exposing more neutralization sensitive epitopes. We knew that we had a protein that would be a good antigen, but as has been demonstrated in this pandemic, making protein in a large enough quantities to vaccinate millions of humans. It's just not feasible in the onset of a pandemic or at least it's not quick. So how do we, again, go back to just the three basic principles. How do we act fast. How do we monopolize on a reliable vaccine technology, and how do we in italicized universal fashion, plug and play what we know into a new platform. And so all of these things came together with our collaboration with Madonna, which is a biotech company that's based out of Cambridge, Massachusetts. So they have a platform by which you can put in just about any new therapeutic protein, make an mRNA sequence here by that mRNA and formulate it into a lipid nanoparticle or a ball of fat. And by delivering that ball of fat that has the mRNA code inside, you can put that into a person's muscle and allow the person's body to act like the vaccine factory. And if you've been vaccinated with the Madonna vaccine candidate and actually even the Pfizer's vaccine candidate, what your body has done is it's made the SARS-CoV-2 spike protein in order to alert your body of a danger to that spike protein so that your body can learn it in very specific details and be able to defend your body against the coronavirus later. So by proof of concept, we then took our MERS-S2P, again the pre-fusion stabilized version of the spike protein, into Madonna's mRNA platform, and asked a simple question, does it protect mice, yes or no, and does it do so in a dose dependent response? And so we were able to use Ralph Barrett's MERS challenge model where unfortunately without vaccine, these animals succumb to the lethal infection of MERS coronavirus challenge. However, when they're vaccinated by 1 or 0.1 micrograms of MERS-S2P mRNA, they are fully protected against weight loss. And even at a very low 0.01 micrograms given per mouse, while the animals are not fully protected, they at least they do recover following day eight in this particular model. What does that look like locally in the lung? Well, it looks like protection from viral load at 1 microgram and 0.1 microgram, and also protection against lung hemorrhage, which is a way to score for the amount of very localized lung disease in these animals. So in all, the MERS-S2P mRNA elicited dose dependent protection against the lethal MERS challenge, and parentheses without evidence of enhanced illness. And while I don't go into that now, at least in the beginning of the pandemic, that was an important point to make as everyone was really concerned based on what we knew about vaccine enhancement from RSV or measles that coronavirus vaccines might be able to do the same or have the same phenomenon. So we had that base of knowledge. And while it only is once live, it certainly was about three years of my life really honing in on the doses really within collaboration with Moderna, figuring out how to best place the spike proteins into their mRNA platform, etc. So we had this base of knowledge. The pandemic started, the sequences came out, and then three days later in collaboration with Moderna, we decided on a sequence for what is the brand name, mRNA 1273. So that is the SARS-CoV-2 spike protein, all 1273 amino acids of the thing, transmembrane anchored. And so we were able to then in collaboration with Moderna, move very quickly towards confirming the immunogenicity in mice. So I usually don't even talk about the mouse studies anymore because actually ironically enough, anything was that was published last summer is now outdated because we're moving so quickly scientifically, which is which is great. But some things that are coming out now in humans were actually confirmed very early on in some of our mouse studies. And so I like to now remind people of that and the utility of what are the small animal models in pandemic preparedness. With that being said, we immunized animals with mRNA 1273 at week zero and week three, and then modestly challenged those animals with a mouse-adapted strain of SARS-CoV-2, going back to Ralph Barrick's laboratory at the University of North Carolina at Chapel Hill. This particular virus has the ability to replicate in wild-type mice, wild-type bouncy mice in the lung and also in the nose. And so we beg the question, how much virus is in the lung after two doses of mRNA 1273? And saw that there was no viral replication at the day of peak viral titer in the lungs of those animals that got one microgram of mRNA 1273, which is actually a very modest dose in a mouse. And then there was a stepwise increase in the amount of virus in the lung. So what that means is we have dose-dependent protection in the same way that we saw it with the MERS-S2P mRNA. And then for the nasal turbinates, there were four animals in the one microgram group and three out of which had no detectable viral titers in their upper airway as well. The reason why I really wanted to add in some of this mouse data is because as we're seeing one-dose vaccines come out and as we are getting kind of what could be considered anecdotal evidence from the Moderna and the Pfizer candidates around one dose of the vaccine being partially protective, I want to remind people that we showed that one dose of mRNA 1273 is protective in mice following after seven weeks even. And that was shown with 10 micrograms and one micrograms in this particular challenge model. We then went on to the non-human primate where we gave a clinically relevant, so the 100 micrograms of mRNA 1273 was then being used or planned to be used in the phase one clinical trial and has now been the dose that it has is under use with the FDA emergency use authorization. We tested that, comparing it to a 10 microgram or a modest dose of mRNA 1273, an obvious PBS group at a zero four week immunization schema, followed by a challenge that was about 10 to the six PFU of SARS-CoV-2 given directly, and also into the nose. And we followed those animals to look at whether or not they were protected in their upper and lower airway, but prior to that, we looked at immune responses. I won't be showing the T cell responses today, but here are the antibody responses in those animals at that week eight time point. So that is right before the animals got challenged and we call it the pre-challenge time point. Firstly, looking at the binding antibodies that are specific to the full length spike protein. You see that there's a dose dependent response. So the 100 microgram group elicits about 10 to the four binding antibodies towards a spike protein and the 10 microgram group elicits about a half log less than that. So there's this beautiful dose dependent response and the clinically relevant dose actually elicits antibodies that are in the upper quartile of what you see in people that had been recovered from COVID-19. So what that suggests is that the vaccine is eliciting just as good and actually better than most people that have gotten COVID-19 previously. We then looked at some neutralizing antibody responses and the story remains the same. We said one thing that I want to point out with the neutralizing antibody responses is that you are as you get into the more functional antibody responses, you're really blowing out what you saw with the people who have been recovered from COVID-19. So we still see this dose dependent response for neutralizing antibodies and this is a pseudovirus neutralization assay, which is standard in our BSL2 laboratory here at the vaccine research center. Here's an ACE2 binding inhibition assay. So how much antibody is there that can inhibit ACE2 from binding to the receptor binding domain. And a lot of virus neutralization assay, all of which tell the same story that we are listening very robust, high level neutralizing antibodies with the clinically relevant 100 microgram dose in the nonhuman primate and as well actually even with a very modest 10 microgram dose in the nonhuman primate. We looked at the clearance of viral replication in both the lower airway and the upper airway. And we did this by way of subgenomic RNAs and determined that, especially in the lower airway, that we were able to basically fully protect those animals with the 100 microgram dose from viral replication and we realized that the virus is cleared very rapidly by day four following the challenge in this Rhesus macaque model. Looking at the upper airway, one of the things that became clear is that with the 100 microgram group, especially that rapid clearance was synonymous to what we saw in the lump. How did that tell us? It told us that the ability to limit viral replication in both the lower and the upper airways had important implications not just for SARS-CoV-2 disease, but also for transmission. The reason why I point that out is because, well, the data from the humans is kind of telling us the same thing at this point, where in the field, as you like to say, or phase four where the vaccine rollout is happening, the Pfizer vaccine has been showing that it is preventing asymptomatic cases in people. And so what that does is it has implications on transmission, similar to what we saw in the nonhuman primate. So it took about four days for Moderna to initiate GMP manufacturing of their clinical grade material. So soon after the day after we decided on the sequences for mRNA-127 degree. And Moderna was able to really rapidly shift that vaccine in 41 days to the clinical trial site, which really goes back to the utility of this very fast and reliable manufacturability of this vaccine concept. We started the phase one clinical trial, and just looking at my time here, I only have about three more minutes. And so that's enough time to just give you a snippet of what happened in the phase one clinical trials. So we started several, the first of which started on March 16 at Kaiser Permanente in Seattle, Washington, led by the great Lisa Jackson. I actually really love Lisa. And then the ages of that particular trial were people who between the ages of 18 to 55, and we assess three different doses of the mRNA-1273. To come on by later in some other clinical phase one clinical trials where people over the age of 56, these colors remain the same on the graph that I will show later. And the data that you will be firstly looking at are data that are taken for antibody responses two weeks following the second dose in the phase one clinical trial. So the first thing that became very clear is that 100 micrograms of mRNA-1273, and the people that were 18 to 55 years old, elicited a 10 to the 3 neutralizing antibody titer in these people. So this is a high level of neutralizing antibody titer from a live virus neutralizing antibody assay. The second thing that was became very clear is that there was a stepwise increase of the neutralization titer. So following one dose of mRNA-1273, we do get a blip of neutralizing antibody responses that really is whopping by about a lot more once you get into your second dose of your vaccine. And especially what was interesting is that in the older people, that number of neutralizing antibodies that is after your second dose of vaccine is on par with what we saw in the younger cohort as well. So that was really, really, really promising to see, especially at the time when it was very clear that older people were more vulnerable to succumb from SARS-CoV-2 virus. And we have since then published out to day 119 in this phase one clinical trial, showing that following that peak of the neutralizing antibody responses, we don't see really any notable decrease in the antibodies following that at least up to day 119. And while we are about a year into the clinical trial, I will say that even just taking peaks, it is clear that the antibodies are at least remaining for some time. I do want to touch on just very quickly the variants, because people are concerned globally by the variant and duly so, right, because it's very scary to note that a virus that is firstly novel in humans is now mutating as well as it transmits to humans. But the one thing that I want to remind people is that number one, that is what viruses do. It is their job and it is our job to not give them our bodies as hosts so that they can do so. Nevertheless, the so-called UK variant was tested against the vaccine seer from the phase one clinical trials that I showed you before. And the conclusion is that we don't really see a decrease in the neutralization capacity against that particular variant. We also tested the so-called South African variant. So this is B1351 where the inclusion of the 484K mutation in the receptor binding domain, this is a mutation that that evades neutralization by very potent receptor binding antibodies. And inclusion of that mutation does take the neutralization capacity of the serum down just a bit. But we don't see complete evasion. So we really still, while there is about six fold decrease in neutralization tighter with the B1351 variant, there's still over 10 to the two neutralization capacity. And reminding people that this level of neutralization is the level of neutralization that is more akin to the homotypic strain of SARS-CoV-2 began. So the inclusion of the G variant actually in the globally increase the neutralization capacity of the vaccines. So we feel like we are good for now, but I definitely want to remind everyone that we would like to see vaccine rollout go very quickly so that we can start to see these variants and currents of new variants appearing across the globe. I will forego the conclusions only to say that mRNA-1273 is currently still in phase 3 efficacy trial and has been granted FDA approval in the US and also approval in multiple countries around the globe. And in the interim analysis of that phase 3 efficacy trial, which was placebo controlled, where 15,000 people were dosed with placebo and another 15,000 were given mRNA-1273. We saw a 94.1% efficacy rate and clear, clear, clear and start efficacy, especially against severe COVID-19. And so I will stop there by way of time only to thank everyone in my lab, especially Dr. Barney Graham, who has allowed me to work on this project for the last several years. And everyone at the Vaccine Research Center who has been so helpful with our COVID-19 response, it's been a VRC wide effort in all. And all of our collaborators and contributors that have contributed since the beginning of this project from the inception of thinking about the spike protein as a vaccine antigen and structural studies and obviously Moderna for their development of this vaccine and for collaborating with us on an academic level, even before the pandemic began. And thank you to the clinical trial volunteers as well. Thank you. That was a wonderful presentation. I think I liked how you really packed a lot of knowledge and how things came together in this field and how all the level of complexity were explained excellently for the amount of time. We already received a lot of questions, but we will have them at the end of the three presentations. So the next presenter up is Florian Kramer from Mount Sinai. So Florian, thank you, and you can get started. Thank you. So I'm going to walk you through what we did here at Mount Sinai in the last 14 months to try to understand the antibody response to SARS-CoV-2 infection and then basically the immune response to vaccination. And I have some new data that I wanted to share that is not out there as published manuscript yet. Just to start out, with a little introduction, I don't have to cover a lot of this because Dr. Krobet has already gone through that. You're familiar with the coronavirus virion structure. We have this RNA genome on the inside covered by nuclear protein. There's a viral envelope with two proteins, the envelope protein and the matrix protein, and then we have this humongous spike protein on the surface. And as Dr. Krobet has explained, this protein is actually used by the virus to bind to ourselves, specifically the receptor binding domain, which is shown here in red on this trimeric structure of the spike is binding to ACE2. And so when the sequence of the new virus was published on January 10th, we started to work on constructs to generate the agents and ACEs to understand the immune response. And actually Dr. Krobet told us how to modify these constructs to get nice expression and to set this up optimally. So we started our work here at Mount Sinai, which is a big hospital complex and of course we had a lot of cases initially. There was not much that could be done to treat these people. And so we set up a plasma program. So we screened people with our essay to look for people who already had zero converted and who could then donate convalescent plasma to treat severely sick patients. Well, there are a lot of questions about the efficacy of convalescent plasma. This helped us to generate a lot of data about the antibody response specifically to mild and asymptomatic SARS-CoV-2 infections. So we have screened more than 100,000 donors and I'm showing you here data from approximately 30,000 positive individuals. This pie chart on the left shows you the data distribution. Our clinical laboratory does categorize dieters in 1 to 80 and 1 to 160, which are considered low dieters, and about 7% of the positive individuals have low dieters. We have 1 to 320, which is an intermediate dieter and about 20% of individuals have that. And we categorize 1 to 960 and 1 to 280 or above as high dieters and about 70% of individuals who were positive had actually high dieters. And here on the right, I'm just showing you people that we screened and then were positive over time from basically April to October. This program is still ongoing. And the blue line here on top indicates the percentage of individuals who had a 1 to 320 dieter or higher. This oscillates around 90%. What I'm trying to tell you here is basically that people have a normal, a robust immune response to the SARS-CoV-2 when they get an infection. Of course, this is a binding essay. We also wanted to see how functional these antibodies are. And so we are doing neutralization essays to look at that with authentic SARS-CoV-2. And here's a correlation analysis on the x-axis. You see binding antibody dieters on the y-axis. You see neutralization dieters. And you can see that there's a very nice correlation, although there is, of course, relatively widespread within these categories of dieters that we have, which goes over at least one log. So there is a lot of variability, but in general there's a nice correlation. And so another important question was how do these antibody dieters develop over time? You might remember initially there were newspaper reports saying that antibodies would go away after eight weeks. And so we looked at that early on and, of course, that doesn't even make sense from a B cell biology point of view. And so we followed people, a cohort of 120 individuals over in the end seven months. And here I'm showing you the days 30, day 82 and day 148 dieters. And you see that initially the dieters are pretty stable and then you have some drop, which is not dramatic. And this actually flattens out from day 148, which is month five to month seven. When we looked at that and stratified it by initial dieter, we see that people who have initially higher dieters drop off a little bit quicker than people who have initially lower dieters, which seem to be pretty stable and have a little bit different kinetics where you have a phase where between day 30 and day 82 you still have an increase in dieter. But basically what we see here is that it seems that this is a normal immune response that produces long-lived antibody responses. And this also holds true when we do neutralization assets. So this is shown here on the left, as I mentioned, there's a lot of variability in neutralizing dieters. But in general, they hold out during the observation period. And even at month five, there is a correlation between binding and neutralizing antibody dieters, suggesting that there's not necessarily a shift in epitopes. So this is good news, but the question that we had back in April was what does all of this mean, right? And so we knew at that point from animal models, and there were very nice papers published by Dan Baruch's group, for example, that infection in non-human primates, and this is shown here on the left, is productive, right? These animals get infected. There's a lot of virus replication in the lung. This is shown here on the left. And then they recover. And then if you try to re-challenge them, these animals are protected from re-challenge. So that's what we knew from animals. And what we also knew from the animal models is that if you vaccinate animals, and this here is also from Dan Baruch's group, where they vaccinated with DNA vaccine that was experimental and wasn't very good, but gave a variety of neutralizing antibody dieters. We saw there that the neutralizing antibody dieters here on the X-axis correlate very well with reduction of virus replication in the lung. So basically, neutralizing antibody dieters correlate with protection, and the data that was needed for protection here wasn't even that high. It was a 1 to 100 dieter, which was good news. So this is basically showing that there is a correlate of protection in the NHB model, and that is neutralizing antibody. Now, we have correlates of protection for many different vaccines and virus infections. I'm just showing a list here from a review that Stan Blotkin wrote. You see we have this for hepatitis A and hepatitis B. We have this for influenza. It's the famous 1 to 40 HI dieter. We have it for measles. There it's a neutralization dieter. And back in April, we wanted to set up a study to see if we can establish a correlate of protection for South Coronavirus to as well. Of course, many different other groups also did that, and some of them already have results. I'm just going to show you a little bit about our study and how that then relates to vaccines. So we set up a study that we called BARIS, Protection Associated with Rapid Immunity to South Coronavirus 2. We enrolled about 400 individuals. Half of them had antibodies already to South Coronavirus 2 from the first wave here in New York, and the other half were negative. And most of them were healthcare workers, so we recruited them in our hospital. And then we do a very extensive follow-up where we take serum and BVMCs. We collect that every two weeks. And we collect the livers so we can actually check for virus for asymptomatic infections. We also check for other infections with a biofire respiratory panel. And we also, of course, if somebody comes in with symptoms, we test the person for SARS-CoV-2 by BCR. And so I'm not ready to share the results in terms of protection. Here's some data on the antibody levels that we see in these individuals. And you can see that overall, these antibody levels stay relatively stable. And of note here, day zero is not the time of infection. It's really the time of recruitment. And for many people, that is one to three months after infection. So they already kind of seem to hit the stable blood though when we enroll them in the study. But the important point here isn't that's what I wanted to point out. These were healthcare workers, right? And in December, the vaccine became available here. And now over 70% of our cohort is vaccinated. And that gave us the opportunity to look at some pretty interesting questions regarding vaccination. And so I wanted to show you two recent findings that we have from that cohort. The first one, and that was a big question that we had was, should individuals who already had a SARS-CoV-2 infection get vaccinated? And if yes, how often? The reason why we asked the question is because the infection initially is like a prime and then you give the first vaccination, which acts like a boost. And so we thought it might not be necessary to give a second vaccine. And so we used our very study to look at people who had no preexisting immunity to the SARS-CoV-2 and then got their SARS-CoV-2 vaccine. And these are the blue dots here were people who already had a SARS-CoV-2 infection and then got the coronavirus vaccine. In this case, it was from Pfizer or from Moderna, so mRNA vaccines. And so this study actually got published today in the Union Journal of Medicine. What we see here is that we have a very rapid response to the first dose of the vaccine in people who were already serum positive. This exceeds by far people who had been naïve previously. And actually the second vaccination doesn't give them much benefit anymore. There's not much increase here anymore between the first dose and the second dose. But these people make an antibody response that really exceeds what you see in previously serum negative individuals, which do mount an antibody response to benefit from the second shot, but they never reach the levels that we actually see in those individuals who had already had SARS-CoV-2 infection. And there's also an important point about the reactogenicity. We know that mRNA vaccines induce quite some reactogenicity, specifically after the second vaccination. And so we looked here after the first vaccination where naïve individuals, the side effects are relatively mild and mostly local at the injection side, but not really systemic. But people who had a SARS-CoV-2 infection then get one shot actually have increased systemic side effects that resemble basically what naïve individuals have after the second shot. And so we think other groups have shown exactly the same now that maybe the policy in the US should be changed from giving the shot twice to already positive individuals to just giving one shot. And a number of European countries have already changed their policies accordingly. Okay, and the other data set that I wanted to discuss is about variants and vaccination. And so what we did here is we looked at the unbiased, plasma-blast response of the mRNA vaccination that I'll get into that. So with our first, and Dr. Krobet already touched on that, there's now a number of variants that are circulating in several countries and increasingly worldwide that are of concern to us. There's three of them that are really problematic. One is P117, which was first detected in the UK, P1351, which was first detected in South Africa, and P1, which was first detected in Brazil. And what stands out with these variants is that they have mutations here in the receptor-binding domain at the interface with the ACE2 receptor, right? We have this N541 mutation, which is shared between all of them. And then the P351 and P1 also have a mutation at position 484 and 417. And there's a lot of focus on these mutations and how these mutations actually change antibody binding and neutralization of the virus and rightfully so. But I also wanted to draw your attention to the N-terminal domain, which is also a target for neutralizing antibodies and which is extensively changed in all three of these variants. And we'll get back to why this is important in a few minutes. So when the first individuals in our various study got vaccinated, we of course wanted to look at their immune response to vaccination. So these are individuals who are previously naive and we can compare them to convalescent individuals that we had, which we categorized into lower responders, medium responders and high responders. And here on the right side you have the antibody response to the spike protein in six vaccinees. And what you can see is that the majority of them really respond very well and their big dieters exceed what we see even in the high convalescent group. So a very beautiful immune response and very high immune response and this looks very good. We also looked at the neutralizing antibody dieters in vaccinated individuals and again we saw that the immune response there was very high. There was a strong neutralizing activity found in their sera, which was in the ballpark of strong, strong responders in the seroconvalescent groups. There wasn't that much of a difference between the high responders and the neutralizing activity at peak in the vaccinated individuals. And that made us curious and recalculated ratios between binding antibody and neutralizing antibody for the vaccinees and also for the convalescent individuals. And we found that actually people who have lower antibody dieters have better ratios between binding and neutralizing antibodies. And the vaccinees, while they had very high absolute antibody dieters, had a ratio that was favoring binding but not neutralizing antibodies. And so we took samples here from three individuals at day 27, which was six days after the second shot. This is when the plasma blast response in blood is the highest and we cloned out unbiased antibodies from these plasma blasts and started to analyze them. And these are the results that I'm showing you here. We had a number of antibodies that were able to isolate here a little bit more than 40 from three different individuals. And I'm showing you here left the binding of these monoclonal antibodies that were derived from these plasma blasts and we have very nice binding to the full-length spike. We see that only a fraction of them bind to the receptor binding domain and approximately as many bind to the end terminal domain. And this is broken down here by the subjects where we see that in most of them it's relatively, or two of them it's relatively balanced. One of them seems to make only entity or full-length spike antibodies but not the RPT antibodies, at least in that small set of monoclonals that we got from that subject. The important point here was also that most of these antibodies are actually not neutralizing and that was surprising but goes well with what we found in serology. Actually, for two of the subjects, we only found one neutralizing antibody. The third subject had a larger proportion of neutralizing antibodies, namely 34. The interesting part here is that the majority of the neutralizing antibodies actually targeted the entity, which I told you is something that isn't really paid too much attention to. And so we wanted to figure out how these antibodies would now deal with variants. At this point in time, we didn't have access to the South African or the Brazilian variant, but we had isolates from Monsignor from a patient that resembled them in a way. We also had this E484K mutation in the RBD in this isolate, which is a key mutation for abrogation of antibody binding to the RBD. And we had changes in the entity including a deletion in a loop here. And what we found was that the RBD antibodies that we isolated, the two neutralizing ones, neutralized this variant actually very well despite the fact that there was an E484K mutation which abrogates binding of a lot of RBD monoclonal antibodies. But what we also saw was that these antibodies that we had isolated that bound to the entity completely lost their activity against this variant. And this is an important point because, again, when we think about neutralization and cross-neutralization of variants, we always talk about the RBD, but we forget about the entity which might be a really important contributor to neutralizing activity. And with that, I'm going to conclude. We saw that humans induce solid antibody responses to SARS-CoV-2 even after mild infection. Antibodies binding to the spike protein correlate with neutralization. The antibody response to infection looks fairly normal. You get an initial strong increase and that is driven by plasma blasts which die after a few days. But the antibodies that they make stick around, they have a half-life of 21 days. Then you see a slow decline. And then the serum antibody level seems to stabilize and this is most likely driven by long-lived plasma cells in the bone marrow. The $1 million question is just, and this is different for every individual, it's this baseline level that you see in an individual above or below the protective threshold. And we still don't have answers for that in terms of a quantitative correlate of protection. We think that for individuals previously infected with SARS-CoV-2, one shot of mRNA vaccine is enough. And we see that the neutralizing antibody response to SARS-CoV-2 is complex and not just focused on the RBD. We would not forget about the NTT and the antibodies that neutralize the virus by binding to that domain. And I just wanted to also mention that the function of that domain is not really understood yet. And with that, I would like to thank my team here at Monsayne and all of our collaborators throughout the Monsayne Health System and at other institutions. Thanks a lot. Thank you, Florian. That was a very interesting presentation and the like how you highlighted a number of other issues that are also important, including the vaccination of people who got COVID previously. Mohamed Sajadi, the University of Maryland, also has data on that. And some of the issues that you... How long is this response going to last? That's a million dollar question, but I always tell people I prefer to have a little bit of immune response and not immune response, I also think. But of course, we like to know that. And I see that there are questions in the Q&A that are asking that, but we'll get to that. Thank you. And last but not least, and in this case, it's definitely true with Rino Rapoli, who is going to present on a different aspect, so vaccine and monoclonal to regain our freedom. Yes, please, you know, give us good news. Thank you. Well, thank you. Thank you, Corbett, and thank you, Florian, for the beautiful presentations. My talk will be a little bit more general, taking a kind of bird eye view to the pandemic. And look at the title of vaccine and monoclonal to regain our freedom. And I guess we'll get back to the last slide to see what I mean by that. But if I can have the next slide. During the last, actually from SARS-CoV-2, from the SARS of 2002, many times when I talk about potential pandemics, I always start from showing a picture of my city, Siena, Italy, Tuscany, where I live. And why? Because in Siena the, I mean, these pandemics are not new. I mean, and to learn more about that, we need to go to the next slide. And that is a painting of Umbrogelor in safety, and it tries to remind what was Siena in 1300, and the 1200 beginning of 1300. Siena was one of the richest cities worldwide at that time. It was located between Rome and Northern Europe. And they took advantage of that because they started to have a very flourishing trade. They set up banks, hospitals, people were stopping, making business, and they became very rich. The city had an incredible amount of people for that time, approximately 100,000 people. Maybe, I mean, we don't know exactly the number, but it was heavily populated. And at that time was bigger than Paris. And as it happens in very rich and wealthy situations, artists were going there, architects, painters, poets, and they were very happy about that, very flourishing thing. And what you see here is the painting of the Scuola Sennese. Scuola Sennese is one of the first signs of civilisation after the Middle Ages, and it's very famous because Duccio de Bernizania, the emperors, basically they did a lot of the paintings which are all over the museums, all over the world. So that was the climate. And in that climate, the Sennese decided they wanted to do something very special. And they wanted to build the largest cathedral ever built. That time building a cathedral was like today building the tallest building worldwide. I mean, you want to show your power. Next slide shows what happened. They started building and they were May 1348. They had built the wall that you see indicated by the red arrow. That was the facade of the cathedral. You see how big it was supposed to be. And this was facing south. The people coming from around would see this immense, huge cathedral and they had to be intimidated. People should know where they were going. But what happened was May 1348, they planned the Black Death arrived to Siena. And in three months from May to September, two thirds of the population died. The economy was collapsed. Nobody stopped there anymore. The cathedral was all the boys, the architects, the painters that died. And nobody knew how to build the cathedral anymore. And basically that was it. The cathedral you see on the back was built later on, but was much smaller than the initial plan. And so that's what a pandemic or an infectious disease can do. Next slide shows the same wall seen from the inside, what this was supposed to be inside the cathedral. And I call this wall with the windows to the sky, basically the largest monument to infectious diseases ever built. And it is there. And I walk through this wall pretty often. And every time that reminds me that infectious disease can come. And in three months can basically kill a lot of people that can ruin an economy, change history forever. And that is basically what has happened in Siena. And obviously why I'm showing that. Well, the question is, what's the difference between Siena 1348 and COVID 2020? Next slide tries to address that. 2020. Well, if I look at how we behave in 2020, I don't think we faced, we did many things which were different from the ones that people did in Siena 700 years ago. I mean, we used quarantine, social distancing, hygiene. The things that we call known pharmaceutical intervention. I mean, those are non-new. Those were available 700 years ago. And those are the tools that we use mostly in 2020 and the tools that still we continue to use. So my comment is that 2020 was middle age. Now, what about 2021? Well, in 2021, and we are seeing from the previous two speakers, obviously we see that science has come in. The activities has been working pretty heavily during 2020 and has been producing mostly two things, vaccines and human morphine antibodies. And now in 2021, we believe we will be able to start to control this pandemic. And obviously one year ago, people were asking to me and many other experts how long it's going to take to make vaccines. So we're saying, well, it's going to take, usually to make vaccines takes 15 years. We'll try to go fast. Maybe we'll do vaccines in 18 months. Maybe in three years. Wow. We did in 10 months. And I think for better really show the beautiful example of that. And so the question is why we made in 10 months? Well, we made for two main reasons. And those are the bottom of the slide. Incredible technology advances and unprecedented investment by the public sector, mostly the American government, but also Europe and UK. Next slide takes a look at the technologies. Well, the bottom of the slide is in 1930, basically says that in vaccination, in vaccines, up to 1930, but also 1960s, vaccination was mostly empirical. You will take bacteria, viruses, parasites, grow and kill them and inject them or attenuate and inject them. Then starting 1980, new technologies can recombine a DNA, glycogonjugation, genomics, reverse vaccinology. And you see on the outer circle, you see the, what I imagine today is that we are living in explosion of technologies. That are really some of which are incredibly important and solve our problems. And each of these new waves of technology solve problems that couldn't be solved before. Now, in the case of COVID-19 vaccines, there are four technologies that really made the difference. One is what I call internet-based vaccines, number one. I'll have one slide dedicated to that. The second is structural vaccinology or structure-based antigen design, which I think for which Corbett gave us a beautiful example of how the spike protein was stabilized. The third one is synthetic biology. I mean, the ability to make synthetic genes in a matter of hours. And four is for other type of arguments of vaccines, both conventional vaccines, the fact that we have licensed adjuvants that can help improve vaccines. So, let's look at what I call internet-based vaccines. Basically, this slide goes back to 2013. 2013 was after the influenza pandemic of 2009. And we are watching carefully globally what was going on with influenza, especially avian influenza. And on Easter day of 2013, Chinese CDC posted on the internet the sequence of a potentially pandemic influenza virus. It was an avian virus that killed three people, age seven and nine, and was supposed to be potentially pandemic. That was the Easter day. On Monday of the same week, Craig Venter had his lab in California, San Diego, basically downloaded the sequence that had been teleported by the internet and made the two synthetic genes of the two antigens for the influenza. On Monday night, he put those two synthetic genes in an envelope and sent them to our laboratories. At that time, I was working for Novartis in Cambridge, Massachusetts, in Boston. Tuesday morning, we started working with those two synthetic genes, and we made two type of vaccines. One was an RNA vaccine, and the other, which was ready in one week, ready to immunize mice at that time. And the other one was we made a real virus, very, very genetic. It was used as a conventional vaccine. And that was the first time that basically we were able to generate one week vaccines without ever seeing the virus. And it was very quick and was using synthetic genes and doing RNA vaccines. Fortunately, that virus did not become a pandemic, but basically with that experience, the world of vaccination somehow, in my mind, had changed. And the way I look at it, the way it changed is shown on the next slide. And basically, I think we have made the transition from what I call analogy vaccines to digital vaccines. What do I mean by that? Well, before we were to make vaccines, we needed the virus, we needed the bacterium, we needed the parasite, then we need to grow, to purify things, to inactivate, to basically attenuate whatever you want to do to make a vaccine. Well, this was the first time that we did not need the virus. We only needed the sequence, the information. We didn't need to ship the virus through customs, through things. It was just very easily teleported through the Internet. And out of that, you make a synthetic gene, out of the synthetic gene, you make a vaccine. So that's, I think, what has been repeated in a very large scale, with much better technologies, in January 2020, when the same Chinese CDC put on the Internet the sequence of SARS-CoV-2. So that's the technology that really allowed to go very fast, and I think corporate provided a beautiful example of how that happened. The other thing that made the huge difference has been the investment. Next slide, please. And this, to understand the importance of investment, I show here the different phases that we have, we make to make a vaccine. Initially, you start on the left of discovery until you get the proof of constant in the lab. Then once you have that, you go into the early development, toxicology, scale lab, GMP manufacturing, phase one clinical trials, phase two clinical trials, until you reach the proof of constant in the clinic. Once you have that, you move to the last part, which is the late development, where you do phase three clinical trials, you do the factory, you do all these things. And overall, these things take a long time and cost more or less a billion. But we had to shorten the timelines, and that was done thanks to the big investment done by the governments. Next slide shows what happened, basically. Well, instead of doing, as you see above, the classical way of doing vaccines, discovery phase one, phase two, phase three, sequentially, which takes 10, 20 years. Basically, we did everything in parallel, discovery phase one, phase two, and phase three. And basically, as soon as the discovery was done, as Corbin said, in a few days, basically you move to start to GMP in phase one, and then as soon as you have 74 days one, you move to phase two, as soon as you have the phase two, you do phase three. And that basically allowed really to make vaccines in 10 months. Now, next slide shows the summary of the vaccines that were made. And basically, it starts again on the left downloading the sequence from the Internet, making a synthetic gene, and then the synthetic gene has been used to make three types of vaccines. I mean, the one on the right, the one above is the RNA vaccines, and Corbin told us very in an elegant way how it was done, how they got to in 66 days to clinical trials. And that was possible because making RNA vaccine is fully synthetic process, basically. You made a synthetic gene, and then your synthetic gene, you make RNA and you inject the RNA. The second one in the middle, you take the viral, the synthetic gene, and you put it into a viral vector, mostly other viruses, but also other viruses. So you splice the gene, the synthetic gene into a viral vector, and then that viral vector is grown and used as a vaccine. That takes a little bit longer, but not much. In three months, people were in clinical trials. And then the next one is the one at the bottom is the more traditional vaccine, basically. The one that is made by expressing the spike protein. Here you have to take the synthetic gene, put it in a cell line, can be mammalian cell, can be insect cell, can be plant cell. You grow the cells, you purify the protein, and once it's purified, you add an adjuvant, and then you formulate the vaccine and takes a little bit longer, but you go to clinical trials that takes more or less six months. So these are the three types of vaccines that have been made. Next slide shows a little bit how they look like. Here, basically, you see at the bottom all the viral vectors with all the companies that made it. In the middle, the RNA vaccines with the companies that made the RNA vaccines. And at the top in green, the protein base plus adjuvant. Now, in order to make an understanding of what I'm trying to say with this slide, you need to look at the blue arrow on the right. That is more or less the span of neutralizing titers in convalescent people. So the bottom line is that all these vaccines induce neutralizing antibodies which are above, which is the blue line below, the minimum titer which is found in convalescent people. And so basically all these vaccines are supposed to give protection. It's true that the viral vector induce lower level of neutralizing antibodies in general. RNA vaccines induce high, in the very high range of the convalescent people. And the protein base plus adjuvant usually induce a little bit higher. Probably now this is where data from phase one probably now, I think the difference between RNA and protein base is not that big as shown in this slide. But the trend I think is correct. So that was the immunogenicity. Now today in the next slide we do have the efficacy and we know the previous one, please. And we have the efficacy and we know that basically the RNA vaccines are 95% efficacious. The protein base or Novavax is 95%. And they brought the viral vectors between the 60 and 90% efficacy. But all of them are efficacious and most important, all of them have a very high efficacy against severe disease and hospitalization. Next slide. Well, as soon as we're happy about this, the variant starts to come. So if you can click quickly through this slide, basically we've got with the 641G and then all the others came in. And as been said before, I mean this creates at least some problems with the immunogenicity. Next slide shows the summary of the data of the RNA vaccines, which basically with the South African variant they do have, I mean, they take a hit of between six and 10, 12, I don't know, times reduction in neutralizing antibodies. Still above neutralization but clearly lower. And so question is how long it's going to last, all this kind of thing. But I think these vaccines are still good, also against the variants. Next slide is a slide taken for a preprint that we published in mid-December basically, where we've done an experiment where we put the virus, the wildfire virus, with the most potent neutralizing serum that we had, and we saw whether the virus was changing. And basically for 40 days, the virus did not do very much, but 45 days started to get reduced neutralization and then stabilized, but at 75 days we got another mutation, reduced neutralization, then another mutation, and finally this virus was able to escape all neutralizing antibodies of very potent neutralizing serum. And as Florian showed, these basically mutations are both in the receptor binding domain and the NTD. So that shows that, I mean, this virus is really able to escape and the dancing between the NTD and the RBD can really find the ways to get around. So we'll need really potent vaccines and ways to stop the circulation of the virus, the variant. Well, next slide. Basically it goes to the other thing, which I want to talk briefly, which are best passive immunization. Passive immunization is now new. The first Nobel Prize for medicine was given to Bering, Emil von Bering, who found a serum against diphtheria, was able to save the lives of people. That was 1890. Then obviously we have not used, we have used a lot of serum for therapy in the past century, but since 1980s we have not been using them too much, mostly for HIV, HIV infections and other things. And early 90s we started to use tumor monoclonal antibodies. And these tumor monoclonal antibodies have been used a lot in cancer, in inflammatory diseases, in autoimmunity, not very much in infectious diseases. But as I will show in the next few slides, in infectious diseases, basically COVID-19 is really probably going to make the difference because these antibodies are really getting in a powerful way also in preventing and curing infectious diseases. Next slide shows one of the reasons why in infectious diseases antibodies are not being used. This shows neutralizing antibodies for HIV. Early 90s on the left B12 was very, very poor neutralizing antibodies. Then with VRCO one 10 years later we got antibodies that were actually 10 times better than the original one. And then with 10-74, which was basically another 10 years later, we got antibodies which are basically 100 times better. And today we have antibodies that are 1000 times. So basically during the last 30 years we improved the way to make very important neutralizing antibodies. So when COVID came, the technology was there ready to go to isolate very important neutralizing antibodies. And that's what the intention was with the red arrow that you see there. Next slide shows what we did in our laboratory. We started to collect blood of people from the Instituto Spalanzana University of Siena. We isolated B-cells, single cell sorting. And we looked at those ones which were basically recognizing the spike protein. Next slide shows what we found. Basically we started from more than 4,000 B-cells specifically for the spike. We found that 453 of them produced antibodies which are neutralizing the virus. But most of them were neutralizing the virus with a medium potency or like 500 nanograms per ml. Some of them were under nanograms per ml. But really we wanted very, very important antibodies that would be able to be given to people low price, possibly not by infusion, but by injection. And eventually we selected the three antibodies indicated by the arrow which were able to neutralize the virus at a concentration of 10 nanograms or below. So next slide shows one of these three antibodies basically works on the left by preventing Amsterdam from being infected, so those were 25 milligrams per kilogram. And if you go the viral type is in the lung, the bottom left, basically you see this complete protection from viruses in the lung. And the same is true for the therapy on the right. Basically here we use four milligrams per kilogram and basically day three the glands are clean and the Amsterdam specifically gain weight again. So the antibody seems to work. Next slide. And we are not the only one who made antibodies. There are many other groups that have been doing some of them will be used for emergency use. But overall I think I'm getting to the end of my talk is that thanks to the vaccines that have been produced, thanks to the new therapy which are the monoclonal antibodies, I think we have the hope to regain our liberty. So why I'm putting there the study of liberty? Well, the reason is that the next slide, I think this virus in addition to devastating the economy in addition to killing more than 2 million more people basically has really taken out our freedom, our freedom to go out, to walk, to work, to travel, to meet friends, to visit relatives, to go to theater, to play sports. I mean really we need back our freedom and hopefully through thanks to the progress the science has made during the 2020. I think in 2021 we can start controlling this pandemic again. With that I want to only show you the next slide, the team that will develop the antibodies in our lab and the next and last slide, all the people that collaborated with us to make this work possible. And with that I really thank you for your attention. Thank you, that was fantastic. Thank you for highlighting the importance of a monoclonal antibody therapy potentially because to this day all the clinicians I talked to say people are not using that therapy enough. It has to be used at the right time but it is an important tool which is underused. So thank you again. There are quite a few questions. I'll try to at least ask a few more before we check out. The first one that I see is vaccine hesitance versus pre-existing condition. Is there any problem with people that have pre-existing condition and get vaccinated? I think I know the answer to that question but let's hear it from Erkis Makia or Florian of both. Well, so I guess we have to define pre-existing conditions because there are several pre-existing conditions. If you define that by what are considered the comorbidities of COVID-19 disease like diabetes or obesity, those types of comorbidities were actually strategically enrolled into the phase three clinical trials at least for the vaccines that are in the US portfolio. And with that, we did not see any severe adverse events related to the vaccine and people with those types of comorbidities and the efficacy of the vaccine remain stable with those types of comorbidities. And so if that's what you're talking about, then you would be fine getting the vaccine in that case. And then each other medical condition really has to be addressed on a very singular basis, preferably via the specialist physician who you work with with that medical condition. Yeah. I don't know Florian, do you have anything to add to that? Yeah, a little bit, but of course I think what Kismika just said is correct. You have to talk to your physician. That's really important. But in general, there's two concerns, right? The first concern is that you do harm with the vaccine. And for example, people who have allergies or people who have autoimmune disease are worried about that. And there have been severe allergic reactions in a very small number of people who got the mRNA vaccines. The rates are 11 per a million for Pfizer and 2.5 per a million for Moderna. So that's very low. And so for these people, I think that the rule of thumb would be, and again, discussed it with your physician, if you are doing well with other vaccines and if other vaccines, other non-life vaccines are not a problem for you, the COVID-19 vaccines are probably also not a problem for you because they're not live virus vaccines. They're in a way similar to other vaccines. If you were doing well with flu shots, with hepatitis shots and so on and so forth, there's not necessarily a problem. The other question that comes up a lot is somebody has a suppressed immune system. Is the vaccine going to work? And so it's hard to address that because there's many different ways of immune suppression, many different degrees. And we can run studies on all of these different modalities. And so what the assumption is that even if a vaccine doesn't work perfectly in a person, it will give you some degree of protection and it's probably worth getting the vaccine. And you can also, at any point in time, check if you actually made an antibody response. That's the easiest. And the antibody response that you see usually correlates also with CD4D cell responses. So if you're unsure if the vaccine will work for you, you can always talk to your physician and get an antibody test afterwards against the spike protein, not the ones against the nuclear protein. So it's a very complex topic and for a lot of patient populations, they're just plainly is no data. And that is kind of the problem. Thank you. The next question is about protection. The person that asked the question is under the impression that the clinical trials were done mostly in white population, which I'm not sure it is correct. And so the question is about protection on non-white. So black population, Asian, I would think the answer is out there, but I'll let the speaker, the experts tell us what they think about protection in non-whites. I mean, maybe I can take that question. The trials that were run in the U.S. were mostly reflective of the U.S. population at the trial sites. So there was a lot of diversity. I think there were also analyses based on the subpopulations, which are often not statistically significant because you basically divide up the trial into different bits and pieces. But I think the efficacy includes all kinds of ethnic groups and all kinds of backgrounds, all kinds of diversity in the U.S. And as you know, for some of these trials, specifically Novavax, J&J, the trials were done at many different geographic sites, including in South Africa and Novavax vaccine, for example, where the J&J vaccines worked very well actually against non-B1351 viruses in South Africa, less so against the B1351 variant, but even against those, they had substantial efficacy. So I don't think that we should be concerned about that. And there were really efforts made to be inclusive and make sure that we get that information for all parts of the population. Thank you. The next question is an interesting one. It's about what progress has been made in identifying correlates of protection for COVID-19? Is work being done to actively identify these correlates? I would think that there will be some part of the question that are obvious, but as I said, let's see if we're almost doing the work. Yeah, of course. I mean, there are several studies that look at that of the natural infection. There's many data sets that are looked at for vaccination, and I expect that in the next couple of weeks we actually should have a relatively firm idea about that. The question is not really about what correlates with protection. We already know that, right? There is large studies like the Siren study in the UK, there was a study in the US, but another large study in the UK that clearly showed that having antibody correlates with protection and gives you protection that's as good as after our vaccine vaccination or even better because they looked at asymptomatic infection too. The question is just, what's that number going to be? What's the equivalence to a flu-A GI type of 1 to 40? We're not arguing anymore about what mechanism, and there are multiple, what mechanism is protective, we're arguing just about quantity. Right, right. In fact, a question that I have, and this for you or for Dr. Corbett or Dr. Apolli, is whether in fact some of the protection that we see may come, and I'm talking also about the variants, maybe some of the protection that we see may come from something else in neutralization, maybe there could be something that also supports the immune system, thinking about ADCC, for example, antibody-mediated cellular taxidotoxicity. Could that be part of something that maybe we should look at? It could and it is looked at, and the immune system has many different ways of protecting. There are very strong studies that show that the passive transfer of serum from one set of non-hemobrimates to another set of non-hemobrimates is actually protective, so it's sufficient for protection to have antibodies. In terms of the neutralizing versus non-neutralizing or other mechanisms that are basically, that antibodies have to protect, it's not gear yet. We know that after the first shot with an mRNA vaccine, your neutralizing data is very low, but you still get protection, and so it could be that there is also, there are also effective functions that contribute to that. Gallit Alpha, for example, has done very nice work on that. There's also work from the Rockefeller group on that with monoclonal antibodies. It's not as gear yet as for other viruses like Ebola or influenza where we know that ADCC, or I shouldn't say ADCC, but FCR interactions play an important role. So that needs to be explored. We'll see. Okay, thank you. Do you find vaccines which have not used the perfusion-stabilized spike protein, which means native spike, concerning with respect to the protection they may confer? I don't know. I would expect, actually, I don't know if all the vaccines that we know are any of them had the non-stabilized spike. I'm not sure about the Oxford vaccine. All the others mostly have one form or another, but I would expect that we'll be inducing more of the binding antibodies and less of the neutralizing antibodies that Florian was showing. I don't know, Florian, what do you think? Yeah, so you're right, Reno. The AstraZeneca vaccine has a wild-tip sequence and, to my knowledge, the Sputnik vaccine and also the Canxino vaccine, a wild-type sequences. The Moderna and the Pfizer vaccine have the two proline stabilizing mutations and the Novavex and J&J vaccines have, in addition to that, the deletion of the polybasic cleavage site, which stabilizes this. Now, in humans, there is no side-by-side comparison, but J&J actually did a very nice study where they did that in non-human primates with all different constructs that had these features or didn't have them. We did something similar in mice and it turns out that removing the polybasic cleavage site and adding the two prolines is actually optimal. If you remove one of those elements, your protection goes down a little bit. Not too much, but there is an impact. But in humans, we don't have a side-by-side comparison. Thank you. I have a couple more questions, probably. It is a question about delay between first and second dose and potentially the interplay between the immune system with the affinity maturation. I can try to start with that and Florian, you can come in. So, usually, for any vaccine, the longer the space between the first dose and the second dose, the better is the response to the second immunization. However, usually we like to have the second immunization as quickly as possible, like 28 days or 21 days, because that basically gives immediately a high protective level. Now, that has nothing to do with... This is a general rule for all vaccines. We don't know for the RNA yet, but we don't have enough experience. But it has nothing to do with the AstraZeneca vaccine. The reason why the AstraZeneca vaccine is recommended to have 12 weeks is because when you give the second dose four weeks, basically you have still antibodies against the virus, against the vector, and the second dose doesn't take. It doesn't work very little. While if you wait two additional months, the antibodies to the adenovirus go down and then you get a little better response to the second dose. Okay. Thank you. This is an interesting question that is about, I guess, the safety. What are the prospects of developing a self-replicating RNA construct to reduce the need for this massive RNA inocular? Well, I can try to take it because our people were the first ones to start using self-improving RNAs back many years ago. At that time, the messenger RNA was basically... That was 2008, 2009. The technology for making messenger RNA was very primitive. In animal models, self-amplifying messenger RNA was by far superior. Now, that was 10, 11 years ago. I think today the technology of the messenger RNA has made a lot of progress and is much better and has shown to be effective in the clean. Unfortunately, the self-amplifying RNA have gone to phase one clinical trials, but we have not seen great results so far. I don't know at this point whether it's because why in animals they are beautiful, they don't work well in humans because they progress in the technology and manufacturing and delivering has not been as good as for messenger RNA. I think we'll need to wait for the publication of some of the data of the self-amplifying messenger RNA before we answer that question. Okay, thank you. Here is a question that is technical. Consider the post-fusion scribe protein. Is the protein conformation obtained once the SARS-CoV-2 is attached to its target human cells or does the term fusion mean something else? I think I can answer that. It's not the attachment. It's post-attachment, right? You have first attachment and then fusion is triggered, right? And then the protein goes in the post-fusion conformation when it actually basically triggers the fusion of the viral and the cellular membrane, right? Or the endosomal membrane. So the attachment alone is not enough yet. The virus attaches and then the fusion step happens. So the final question. What would be the effect of receiving multiple vaccines? So you get a shot with AstraZeneca and one with Pfizer. I mean, in research, that's used a lot, right? We know from influenza that that works well. We know from HIV to a certain extent that that works well. It's a strategy that is used quite often and giving two different technologies often better than giving the same technology twice. Of course, I will not recommend to do that right now outside of a clinical trial, but there are several clinical trials that are being set up or actually looking at that for SARS-CoV-2 vaccines. And I guess we'll have to wait the results, but I expect that the immune response in these cases would be quite substantial and maybe better than the vaccines that are tested in combination on their own. Yeah, I wonder because at least... I hear stories at least, for example, in Maryland, apparently the pharmacies have waiting lists and you go on the waiting list and they call you and say, if you can come here within an hour, give it a shot because somebody did not show up. So I wonder if there would be an experiment on going just because these things are happening, that you may get a shot one day because the pharmacy called you and then the other day the pharmacy calls you and you get the booster shot that is different from... I don't know how they will deal with that. Yeah, that should not happen right now. I mean, of course, they will call you if you're on the waiting list, but they shouldn't give you different shots. Okay, hopefully they don't. Yeah, if you receive the Pfizer vaccine, you should receive the second dose from Pfizer as well. That's the guideline right now. And as long as there is no change in guidelines, you should probably follow what the guidelines are. I like that. I like the idea to follow. Yes, okay, I know that there are more questions, but I think you have been very generous with your time. So I'm just going to... Unless there are any other consideration from the FEMS people, I would like to thank you once more. Thank you also the other speakers that have left. And thank you. I think it was a fantastic experience from my perspective. It was an informative webinar and it was really important to have it. It will be made available online, so people will be able to listen to it even after the fact. Okay, so thank you very much. Thank you. Bye-bye.