Intro to Viruses, Antivirals, and Vaccines – Dr. Pamela Bjorkman

What I would like to do is introduce you to viruses in general and the corona virus that we’re all worried about in particular. So I’m going to be doing this with reference to my own lab’s work on HIV-1 because our research into corona viruses is more recen. Okay so first I wanted to get some nomenclature down Covid-19 is actually the disease caused by the current corona virus which is called SARS-CoV-2 so you can see in this view this is a schematic of the virus but this is actually an electron micrograph so this is more accurate The corona virus family of viruses in general were named for the fact that they have these spikes that stick out. A lot of viruses have those so that’s nothing new but the corona virus spikes are really large and they create this corona like a fact or a crown so it was named after that. Now I want to say some caveats: the first is in my personal opinion I would recommend that you get information about Covid-19 in this particular virus from scientific websites or from the Center for Disease Control or the World Health Organization or preferably from your doctor. I don’t recommend new sites. I’ve seen some of the science is incorrect on several news sites I’ve looked at. And I also want to say I’m not an MD so I’m going to concentrate on the science, which I’m trying very hard to get correct. Okay so first I wanted to say that of course the world has experienced pandemics over the years as you’re well aware and I want it to highlight from this nice graphic here – what they’re doing is showing you in proportion the death toll for these various pandemics from highest to lowest And so what you can see is that the bubonic plague was the worst that the world has experienced at least in recorded history, but there were others that were quite bad as well. And I wanted to highlight smallpox. I’m going to be saying something about possibly reusing the smallpox virus as a vaccine vector for Covid-19 disease. Also as you’ve all heard about the 1918 flu killed a lot of people around the world and then is still ongoing. HIV that causes AIDS has killed probably up to thirty five million people since that pandemic started in the 1980s and then here we are with Covid-19. This graphic unfortunately is out of date so more people have died than this. It might not look like much but unfortunately I’m afraid that death toll is going to get higher. And then MERS and SARS are caused by corona viruses that are related to SARS Kobe – the virus that’s causing Covid-19. This is Middle East respiratory syndrome and severe acute respiratory syndrome these have pretty much they’re not happening anymore in the world at least MERS isn’t but and SARS may be a few cases So I wanted to mention these because on the next slide you’ll see that SARS MERS and SARS Kobe – are related to each other they descended from bats and that’s common for corona viruses in general. So the initial host of these viruses were bats which harbor many different corona viruses and these sometimes pass into humans in particular they often pass through intermediary hosts. And so the intermediary host that passed for human into humans was probably a civet or a raccoon dog For SARS for MERS it was camels and for SARS Kobe – it might be a penguin but people aren’t really sure right now. But eventually these change enough so that they’re able to infect human cells. I want to point out here that these viruses here which are also corona viruses caused common colds. So these four right here these two into humans maybe hundreds of years ago, possibly even a thousand years ago, and now they’ve attenuated to the point that they caused a lot of the colds that children get and adults get as well So, about the biology of the iris coronavirus – it’s an enveloped virus and that means that it has a lipid membrane around it which is shown right here in this picture. It’s this red part And so this means that the virus itself acquired that membrane from the host cell so that’s something that the virus picked up from the host cell it also has genetic material which in the case viruses can be can include either DNA or RNA where DNA is the code and RNA is the

message that allows you to actually make proteins. So this is an RNA virus and then I want to point out that the spike protein which gives it its coronavirus name. It’s a very large protein so that being said this is all in common with HIV-1 and many other viruses HIV-1 happens to also be an envelope virus. It’s an RNA virus and it has spikes on its surface. There are a lot of differences between the two and HIV-1 is not a corona virus, but what I want to say is that the purpose of any virus including SARS-CoV-2 is to get into a cell, make copies of itself and then get out and spread to other hosts And so a virus is really a parasite it doesn’t include enough of its own machinery to be self-sufficient. So you can’t describe a virus as being alive, whereas a bacterium for example is self-sufficient and it can grow on its own A virus needs a host cell and in the case of SARS-CoV-2 it has adapted so that it can use human cells as its host cell. So that’s the purpose of a virus – get in, hijack the cell machinery, make more copies of yourself, and then transmit to other new hosts so that they can make more copies. So it’s a hijacker and of course that’s what HIV does. It gets in, makes more copies of itself and then gets out. But I want to point out that one of the things my lab studies for HIV and that we’ll be studying for SARS-CoV-2 as well is how it gets in and in the case of HIV it’s been known for decades now that what happens is that the the spike on its surface which is a trimer it’s depicted over here the SARS Coby – spike is also a trimer meaning it has three identical subunits its receptor is a host protein called cd4 that makes the HIV-1 tropism limited to cells that express cd4. And these are cells in your immune system so when HIV infects you you wipe out the cells in your immune system and then your immune system doesn’t really work anymore and you’re susceptible to all kinds of opportunistic infections which leads to AIDS-acquired immunodeficiency syndrome. Okay so for the corona virus life cycle the receptor is not cd4. It was discovered incredibly quickly by researchers that since this came out in January or so the world became more aware of it its receptor is a protein called ace 2 which is on the surface of, for example, lung cells and it’s in other places in your body as well. So what has been discovered also in remarkably short time is that the spike protein, when it interacts with ace 2 in order to actually enter the cell, it needs to be cleaved by this protease – which is called a protease as a enzyme that cuts other proteins that’s what these scissors mean – that protease is called TMP RSS. Now notice this paper from David Villiers group at University of Washington, this is from 2017 so it couldn’t have been done for SARS Coby – it was actually something that was known for SARS and some common cold corona viruses but it turns out a number of these use the ACE 2 receptor. So let’s talk about ace 2. As I said before, ace2 is used as a receptor for SARS CoV2 enter human cells but SARS also uses it and so does at least one of the common cold corona viruses. So ace 2 means angiotensin-converting enzyme 2. What that means is that it cleaves angiotensin, and that’s a peptide hormone that controls basic constriction and blood pressure. More about that later but let me just say that it’s known from previous research that ace 2 is on the membranes of cells that are in the lungs, the arteries, the heart, the kidney, and the intestines. So you may have been reading that there are often GI problems in Covid-19 disease. There can be heart problems, kidney problems and so on And then where the receptor is expressed what types of cells that can determine you know what goes wrong in thisCovid-19 disease. Okay so the fact is that ace2 is important in controlling blood pressure, and so the interaction has been people have been worried about perhaps taking ace inhibitors if you have high

blood pressure. And I just wanted to say that the American Heart Association has said unequivocably: Do NOT stop taking ace inhibitors if you are currently taking them. The reason I’m mentioning this is that they may have effects such that they lower they lower or raise the levels of the ACE 2 receptor so they affect the levels of the ACE 2 receptor on the surface of their target cells and that could contribute to lung damage. So people were somewhat worried about taking these inhibitors and the doctors are telling you do not stop and please talk to your doctor about this if you’re considering it. On the right what you’re seeing is something from a structure of it’s a 3-dimensional structure – this is what people do to visualize interactions at the atomic level between two different proteins. This is actually a structure that was solved 15 years ago now for another coronavirus. It was actually the SARS coronavirus and this is the receptor binding domain of that of that of SARS. Actually in the cyan color there’s a receptor binding motif here and then this is the structure of ace2 and that was solved by a group at Harvard 15 years ago. So now this is available for SARS Coby – it actually looks very similar so people are trying to figure out differences between those at an atomic level right now Okay so back to the HIV lifecycle What we’re going to talk about now is something general for all viruses they have to make copies of themselves so that’s step 2 so I wanted to point out that there are enzymes that do this and the enzyme that does it for SAR – sorry for HIV-1 – is something called reverse transcriptase this makes copies of the HIV viral genome. Now a little aside here: HIV is an RNA virus but it makes copies into DNA, which is the message and then it goes in and it does something really terrible because it’s what’s called a retrovirus. It puts the DNA copy into the nucleus of the cell. It actually integrates it into the chromosome. And what that means is the cell can, you know, not even be producing virus but it’s got a copy of the viral genome So at any time in an infant in an HIV infection the cell can start making new viruses so you cannot get rid of an HIV infection with antiviral drugs because it is integrated in its its genetic materials integrated into your own cells And this is what’s really horrible about this type of virus – which is called a retrovirus. And as many of you may know the discovery of reverse transcriptase was done by David Baltimore. It was a long time ago I believe I forget what year but David was 37 years old when he got the Nobel Prize for this discovery. The reason it was really an amazing discovery was that at the time the central dogma of biology was DNA makes RNA makes protein so the idea was genetic flow of information would only go from DNA into RNA and then RNA would give you the information to make the protein and the protein would do the business. This was reversing that flow of information: RNA going back to make DNA And he discovered this for a different retrovirus than than HIV but it turns out to be what HIV also does. Okay back to the point about reverse transcriptase It makes a huge number of errors, this results in a enormous number of viral strains even within a single infected individual and this is a problem, a horrible problem, for HIV because that means that it’s very hard to make a vaccine. It’s very hard to make antibodies or protective immune responses against so many different strains inside you. Now fortunately, for the corona viruses they actually don’t make that many errors. And the point I made here was that the HIV reverse transcriptase, which we would call an RNA dependent DNA polymerase, has no proofreading activity. So things that copy DNA, like in our own bodies, it’s really important for them to not make mistakes or these would result in mutations. Mutations are not good for a single individual usually, so we have proofreading activities that fix those mistakes. This virus has no proofreading activity. In fact as I’m going to tell you, corona viruses do and they are

unique in RNA viruses and having that sort of proofreading activity. And if you think about it it’s in HIV. HIV, if I can anthropomorphize here, wants to make that many mistakes. It WANTS to make a lot of different viruses and then it is survival of the fittest. It makes all kinds of viruses. Most of them don’t work well, but the ones that do go on to make new strains and then your immune system is all confused. So back to the corona virus – now the corona virus only copies its RNA message into RNA so it doesn’t have a DNA form so it is not a retrovirus. So it has what’s called an RNA dependent RNA polymerase and that’s associated with a proofreading enzyme That’s unique among RNA viruses, to my knowledge. Okay so that’s going to be important for thinking about possible drugs that you could use to combat corona viruses. So speaking about drugs, I want to illustrate that although it’s hard to make antiviral drugs, there are very successful antiviral drugs for HIV. So those have been developed over the years. There are many different inhibitors of reverse transcriptase the enzyme that copies RNA into DNA these are just analogs of the building blocks of the nucleic acid. So these are what are called nucleoside analogs. These are building blocks related to building blocks of the the DNA. So many of these exist. And then there are inhibitors of integrating the RNA into the nucleus, into the DNA. There are then protease inhibitors. I’m going to come back to this later because this is the thought that you could inhibit a viral protease is being considered for SARS-CoV-2. So often viruses require their own protease to cut up their own proteins – and this is an absolute requirement for HIV it cannot survive unless it has its protease – so there are inhibitors against the HIV protease and then there are inhibitors that stop fusion from taking place, which then means the virus can’t enter. Now the important point about the HIV drugs is you must give at least three at a time because HIV mutates so fast that if you give it just one drug it will mutate to become resistant to it so if you give it three or four drugs it can’t necessarily mutate fast enough. And as you may know in this antiviral therapy for HIV/AIDS was available to people starting in 1996 and it was pioneered by David Howe who’s a Caltech alum, and I think still a trustee at Caltech. He was Time Magazine’s Man of the Year for this I think it was that was in 1996. Okay, so you have to give three at once because of the very high mutation rate of HIV. I think it might not be a bad idea to give several at once for the corona virus. It also mutates. It doesn’t mutate as much because because of this proofreading activity of its enzyme. Okay so now I want to talk about antiviral drugs that are in development now that might help control the Covid-19 disease. The great thing is that some of these are already FDA-approved for other other illnesses and so they’ve been tested in humans. They know about its efficacy and safety for these other diseases that they’re being used for and so these could be ready and some of them are even in people now. So that’s a great thing is that if we can take already approved drugs and use them there’s a hope of really quickly going after this disease. Okay so I wanted to mention several places in the corona virus life cycle and in particular the SARS-CoV-2 life cycle where the drugs have been, are being tested. The first of these is an inhibitor. Remember or I said there was a protease called TMP RSS this is actually at the surface of the cell and it is a host protease. The host makes this but it turns out that actually this spike protein has to be cleaved before it becomes active. So if you inhibit TMP RSS, it appears, at least in vitro, that this virus can’t enter as well. So that’s one thing that’s already approved drug so that could be used perhaps in

treating Covid-19 disease now. There’s enough protease that’s in an intra cellular organelle called the Golgi apparatus This is where proteins pass through on during their biosynthesis. That enzyme is called furin and there’s an interesting thing about SARS-CoV-2 spike compared to the sour spike and it appears to be cleaved by furin to activate it on its way out and so people are trying to develop anti furin reagents. Now I’m sure you’ve all heard of chloroquine and hydroxychloroquine. I want to discuss another point of the SARS-CoV-2 lifecycle which involves going into these compartments inside the cell that are called endosomes and these are actually acidic so I’m pointing them out here and in this very busy slide that shows you the life cycle of SARS, sorry, corona viruses in general. This was of course from a 2019 article published in the spring so they didn’t know about SARS-CoV-2 at the time but they were pointing out that corona viruses go in into this low pH compartment called an endosome. So it’s important, the pH is low, so at least from the research I’ve read SARS Co v2 probably also enters and assumes – at least I’ve seen some evidence that says it does in in vitro experiments. This might be relevant to why weak bases such as chloroquine and hydrochloric on these are currently licensed for use as anti-malarial drugs. Also as I’m going to say in a moment for some autoimmune diseases so these are already licensed so this could be an off-target or an off-label use of it for SARS Co v2 infection. Okay so if you raise the intracellular pH of this some of the enzymes that are critical in here probably don’t work but that might not be the whole story or it might not even be any of the story. If chloroquine and hydroxychloroquine actually are efficacious they might be doing this by just in general reducing inflammation. These drugs are being used in patients with autoimmune disease such as rheumatoid arthritis or lupus, and so there’s some thought that the pathology related to Covid-19 disease when people get what’s called acute respiratory disease syndrome or ARDS, is that your immune system is your immune system in general you know does all these things when you’re infected with a virus which are trying to get rid of the virus and one of them is to give you a fever to produce more mucus to make you cough and sneeze that’s the idea is to make you expel whatever In fact sometimes it gives you.. if it’s a gastrointestinal pathogen it will give you diarrhea or make you vomit to get rid of the agent. So there are all these things that happen that the pathogen actually doesn’t do, it’s your own immune system. But it’s there to protect you. Well unfortunately it appears to go quite off in when people have a very severe reaction to SARS-CoV-2 infection. As you’ve heard, in about 20% of the cases it leads to this acute respiratory disease syndrome. And the people who are dying have a horrible reaction from their own immune system. And so the idea is that you could possibly reduce inflammation and that’s why they’re also trying to use an existing drug against one of these. It’s called a cytokine that creates.. it’s produced by your immune system and it creates inflammatory events. It’s called il-6 interleukin 6 and so a company has made an antibody against il-6 that’s used for autoimmune diseases that’s being tested for Covid-19 treatment. And from what I’ve heard it might be more efficacious if you first put the person who’s in severe distress on a steroid – on some prednisone or another steroid that also would reduce inflammation I don’t know if the story is out on that, and all of these have to be done by real clinical trials. But anecdotally I think I’ve heard interviews with doctors in New York that say this may be a promising treatment. Now another potential drug target is the viral protease itself. This is not a host protease, so not to confuse this with what I was talking about. TMP RSS and also furin – these are in the host cell

They’re there to cleave their own proteins but they also cleave the SARS-CoV-2 spike. But there’s a viral protease as well and just like in the case of HIV-1 infection.. One of the drugs that’s used to treat HIV-1 infected people is a protease inhibitor this was developed a long time ago now in the 90s. There are a series of these but now some of these HIV protease inhibitors, these are being tested for whether or not they might work to inhibit the SARS-CoV-2 protease. So this requires a lot more testing. You can actually try and see if you can predict what protease inhibitors that are already approved might work if you have actually the real structure of the viral protease and then you can do drug design to try and see if you could fit in known inhibitors. And so on, and then test these in the lab and then later in clinical trials. Okay now there’s another thing that your body is trying to do to prevent infection by this virus As soon as you get infected by a virus you go through first what’s called an innate immune response, where you just you don’t recognize the particular virus itself but you see that there’s something weird going on in cells because there’s a lot of viral nucleic acid that shouldn’t be there. And that triggers what’s called the innate immune response. It’s called innate because we share a lot of the components of this with invertebrates such as, you know, flies or worms or so on This is distinct from what we call the adaptive immune response which is specific for a particular pathogen Now I’m talking about antibodies and I’m also talking about a type of lymphocyte called a T lymphocyte. I’m not going to really talk about t-cells here, I’m going to concentrate on antibodies. So if your body can make antibodies, it takes a while. It takes five to seven to ten days for people to do what’s called sera converting, which means that detectable in their blood there are antibodies to SARS-CoV-2 and during that five to ten days you need to have some kind of immune protection so at least you have the innate immune reaction. Now, the adaptive immune response is specific to vertebrates so that means, you know, birds, mammals, us, mice, and so on okay so we can all make where we can make antibodies. Some of these are what are called neutralizing antibodies and that means that they block the interaction between the viral spike protein and its receptor. And if you block that, the virus can’t get in so none of the rest of these things happen Which would be great. So let me tell you a little bit more about antibodies. These are proteins in your blood They combat pathogens. This is something that, as I said, is part of the adaptive immune response, and these antibodies through an amazing recombination at the DNA level, allow you to make vast numbers of different types of antibodies. So if there’s a new pathogen that has never been seen before on earth you can make an antibody against it through this amazing way that vertebrates specifically have Let’s talk about us, humans, or the mouse model system. We can make at least 10 to the 16 different types of antibodies. We don’t make all of them at once, but if we need to we can make Nobody’s against anything okay so that’s being depicted here by different colors of antibodies. So as you may have heard there are two ways people are talking about using antibodies to combat Covid-19 and the general principle is called passive immunotherapy. This means that rather than wait for your own immune response to create an effective neutralizing antibody response which might take too long to help, you give them antibodies from someone else who has recovered from the disease. And so actually this passive immunotherapy has been done for, you know, for a long time Actually if you have a rabies virus, if you’re bitten by something that’s rabid, and you have to have a rabies treatment, part of it is passive immunity. Okay so this is not a new idea But how it would work for Covid-19 disease is that you could perhaps get

well, and this has been done in some cases. You get convalescent serum from people who’ve recovered from Covid-19, and this contains what I’ll call a polyclonal mixture of antibodies against SARS Cova. And the reason is polyclonal is when you make an antibody against a virus or anything else, you make lots of different antibodies against it, you don’t make just one kind. So you make this – it’s called a polyclonal response – so that’s illustrated here by lots of different types of antibodies. Okay the other thing you can do is you can take the serum or plasma from a patient and you can actually isolate defined antibodies. And if you isolated to find an antibody that’s called a monoclonal antibody as opposed to a polyclonal antibodies. So let’s say this person had made all these different antibodies here and a researcher got the plasma from that person – they can isolate cells that would make all these different antibodies, do single cell sequencing of those cells (they’re called B cells or B lymphocytes), get the genetic sequence of a particular antibody, and then in the lab that can be produced as a recombinant protein This is how in some cases antibodies are identified, in particular viral diseases So you might decide, you might isolate it, all these different antibodies here, and you test them in vitro and you decide that maybe this pink one is the best one But you could also produce, you know, the purple one or the brown one, whatever And you could give it as a mixture, but this is a defined way of doing this. There are advantages and disadvantages of both of these. This can be done right now from convalescent serum. The problem is you can’t drain the person completely and there just isn’t enough convalescent serum to do this. In the long run it might be better to do individual antibodies or mixtures of individual antibodies, but that’s gonna take a while. Now we’ve been collaborating with Michel Nussenzweig’s lab at Rockefeller University for at least 10 years to actually do this for anti-HIV antibodies. They do single cell b-cell cloning to isolate particular antibodies against – for example we’ve been doing this for HIV-1 and also Zika and we also have a ongoing effort to look at these antibodies against hepatitis C virus and so on. Okay, but the class we’re engaged in a collaboration now with the Nussenzweig lab for them to isolate and antibodies against SARS Kobe – and then we would look at them bound to the SARS COV-2 protein structurally. And so let me just show you what that would look like in the case of HIV-1. So this is an example of something we’re doing for HIV-1. This is a structure and these are we have resolved this to the level of individual atoms and we’re looking at two different antibodies. It doesn’t matter their names but their names are over here and these are bound to the HIV spiked protein. And what you’re seeing now is the distribution of carbohydrates on that protein because it tries to hide most of its surface with carbohydrates. Those carbohydrates are put on by the host machinery I’ll just run that again. It’s trying to hide its protein surface so that antibodies can’t react against it That is very common trick of viruses, SARS-CoV-2 and SARS and so on do that as well. Anyway, so once we know where the antibodies bind we can understand how they bind, which ones are good, which ones are not so good, and so on. Now the other thing we can do is we can note that if we want to make a vaccine by for example, let’s say injecting this particular HIV spike protein, there are parts of it that produce what we call distracting epitopes. These are parts of the protein that are exposed, but they’re not conserved from one strain to the other Of course antibodies target these and then these are not useful because they only work against certain strains so I’m saying this because this gives you a context then of what we’re trying to do for HIV-1 and now we’re trying to do it for SARS-CoV-2. By having these structures we can either engineer them using protein design to make them more efficacious, and we’re also engineering imagens that you could actually inject into eventually maybe as

a vaccine to elicit neutralizing antibodies. These have to be so-called broadly neutralizing antibodies, in the case of HIV-1, because there’s so many different strains. And here I want to point out that these sorts of structural studies are only possible at Caltech through support of facilities, for example the Caltech Molecular Observatory, which is a facility that allows researchers at Caltech to solve structures by x-ray crystallography and the Caltech Cryo-EM Center, which allows researchers at Caltech to solve by a method called cryo-electron microscopy. So without the support of these kinds of basic research facilities at Caltech, my research and the research of other structural biologists at Caltech would not be possible Let’s talk a little bit more about neutralizing antibodies. I showed you that what happens for SARS-CoV-2. It also happens for HIV. If you have a neutralizing antibody and blocks entry and then nothing else happens. So, this is great. People raised these when they’re infected with HIV, but in the case of HIV, most neutralizing antibodies neutralized only a subset of HIV-1 strains and this isn’t going to work because in a single infected person there are more strains of HIV-1 than there are influenza strains in the entire world. So this isn’t going to work. People raise these antibodies, it’s not going to work but we’re hoping that if we engineered them perhaps we could get them to be so-called broadly neutralizing antibodies. So for HIV-1 we need them to be broadly neutralizing antibodies This leads us to the question about, well as you’ve probably read, SARS-CoV-2 also mutates so can it make mutations that would confer resistance to antibodies Yess it can but compared with influenza, which as you know is a virus that comes in a lot of different strains SARS-CoV-2 exhibits relatively few mutations. So let me just say first that we have influenza vaccines, but they have to be changed every year Researchers go around, they identify the three or four strains that they think are going to be most prevalent in the world, and they make a vaccine in a variety of ways against it. That’s why you have to keep repeating your influenza vaccines every year. Okay so flu has a relatively high mutation frequency, but it’s nowhere near as high as HIV-1 Look at SARS-CoV-2. Now what you need to look at here is not the slope of these lines, because the y-axis is different in all cases. What you need to look at is these bottom numbers here: how many mistakes or mutations are made on average per 10,000 units, which in the case of this is a unit of the genetic message or the the nucleic acid Okay, so flu is up at 35 per 10,000 SARS and measles are actually quite similar So let me tell you something about measles. This is this anecdote which is really instructive. So there’s something called mu logical memory and this is the basis of why there are certain types of viral diseases you can get only once Chickenpox is a good example, measles is another. There’s something called immunological memory, so when you make an antibody response in some cases you make what are called memory b-cells and they hang around for a long time and then if you get reexposed you can immediately make a immune response or you can more quickly make an immune response. So if it would normally take you five to seven days, you might have died by that time. But if you have memory b-cells, you would you would do it a lot faster and it would confer protection. So here’s an an anecdote that shows you how that can work in the best possible case So in 1781, these sailors came to the Faroe Islands and they were infected with measles. And the people from the Faroe Islands got sick and a lot of them died. Then in 1846.. they had had no contact with measles between 1781 and 1846. At that time another ship comes in: more measles infected sailors. But people who were old enough to have gotten the measles and survived from the 1781 infection, they didn’t get measles because they had lifelong immunity to measles virus This is incredible. We make a really great response to measles. We don’t have

immunological memory for some other viruses that last as long. So for example from what’s known for the common cold coronaviruses, probably the immunological memory is shorter, it might only be a year. But it’s still – there is some immunological memory That’s what gives us hope for making a vaccine. It might have to be, you’d have to figure out how often to administer it, but I think there’s hope for making a corona virus vaccine. It just may not be work as well as the measles vaccine, which will give you lifelong protection Okay so what about a vaccine? I’ve sort of alluded to this but most current vaccines work by inducing antibodies against a virus: You inject something into what’s called a naive individual – that is they haven’t been exposed to that virus or maybe it’s a bacterium – then the immune response makes antibodies to it. Some of these go into these memory b-cells and then if you’re exposed to it later you’re protected more quickly. So, that’s how it’s thought that most vaccines work and it’s actually more complicated than that as I’ve done more reading. I have to say, the immune response to viruses is extremely complicated. We’re just learning more and more about it all the time. There needs to be a lot more basic research to understand how our natural antibody response and our natural immune response works for viruses. But in the case of some vaccines, it’s believed that antibodies are the so-called correlates of protection. So the question is, you give a vaccine or you get infected yourself and if you’re protected against a reexposure to that – what is protecting you? And it’s thought that antibodies are often a correlative protection What about a vaccine for SARS CoV-2? Let’s go back and think about vaccines in general. In the US and Western countries, we think about vaccines as invented by Edward Jenner. He noticed that milkmaids who were exposed to cowpox from milking cows didn’t seem to get smallpox – which as I showed you in the beginning one of the beginning slides was an enormous problem hundreds of years ago. So he had the idea that “well, let’s just inject people with cow pox.” That would be an example of what’s called a live virus. It can replicate but it’s attenuated so it gives you less of the disease or maybe no disease at all. Okay so that seemed to work That’s attributed as the first vaccine Just as an aside, he later experiments on other children, including his son These sorts of experiments are obviously illegal today. But actually immunizations against smallpox were introduced over a thousand years ago through a process called variolation In places like China they would introduce dried smallpox scabs into the nose of someone who hadn’t been infected and that person would get a milder form of the disease but then would be immune to smallpox. It wasn’t perfect, so one to two percent died after variolation, but that was compared to 30 percent who died from smallpox. Okay so what about a vaccine for SARS CoV-2? Well, we don’t have one yet, but I think it’s kind of promising that they were able to make – – researchers were able to make a vaccine against MERS coronavirus. Remember this is the one that.. it’s not infecting people anywhere right now.. the intermediary host it went from bats into camels. And this was a vaccine for camels. So they made this vaccine, and this picture from the Journal article shows that they were assessing how much mucus was formed by the camels. I didn’t actually know they developed mucus in their nose but apparently they do. So they claimed that this was successful in the camels People aren’t trying to develop MERS or SARS vaccines these days because these viruses are not a problem for the World Health these days. But what this was was – it’s worth looking at this – it was actually something related They used an attenuated virus, meaning it’s not going to make the camels sick. But it was actually the vector for it, so that’s a term that we use for how we might insert MERS proteins. It was a derivative of the smallpox vaccine. So the virus is actually called vaccinia. It’s used meaning it’s used for vaccines, and they just put some genes for MERS into the

vaccinia virus they they changed so it couldn’t replicate anymore. And that was used as a so-called vector for a vaccine for injecting into these camels So this pox vector or vaccinia virus can be used for non-pox-related diseases, which I’ll come back to when I talk about SAR potential SARS CoV-2 vaccines Okay so that leads me to believe that there’s some hope for making a vaccine At least in this way so I want to share with you a PDF that I found from the W.H.O It’s a draft landscape of the potential candidate vaccines, and this was accurate as of the 21st of March But there’s even more now. They had a disclaimer on their website – they don’t necessarily advocate any of these and they’re just telling you what’s out there. So I wanted to put their disclaimer here on this slide. So there’s two, at least as of today I believe, it could change tomorrow, but there are at least two that are in clinical evaluation. That means in people One of them was made in China by the Beijing Institute of biotechnology in a company called Cansino. It’s in Phase I clinical trials for just safety right now, and it was based on using a vector from adenovirus. So it’s not a pox vector, it’s a different type of virus that sometimes used for these types of vectors for vaccines A previous thing that was very similar was tried for Ebola and then you may have heard about US company called Moderna. They’re collaborating with the National Institutes of Health this is National Institutes of Allergy and Infectious Disease, which is what Tony Fauci heads up They’re collaborating with the NIH and they’ve made what is called an mRNA based vaccine. It’s put in lipid nanoparticles. That’s what LNP means – it just means they put the genetic message encoding. They don’t tell you this here, but it encodes the SARS CoV-2 spike protein. That’s what’s necessary to get into a host cell. And so the idea is the message would go into your own cells. Your own cells would make the proteins. All kinds of immune reactions would happen, and hopefully you do all kinds of stuff including making neutralizing antibodies that would prevent interaction of the SARS CoV-2 to spike protein with its ace 2 receptor This company has done this for multiple other things. I believe they’re trying this for influenza virus. This is a very fast way of doing it because it’s very easy for them to make messenger RNAs They have to alter them a little bit so they can’t be cleaved in vivo, but normally they’re very sensitive to getting cut up and stuff It’s pretty fast, I guess, to produce sufficient quantities of messenger RNA It’s quite hard to produce very large quantities of the proteins that the messenger RNA encodes, if you wanted to inject that. So that’s why they were able to do this so quickly. I’ll just go through very quickly – there are other companies that are trying to do this with DNA instead of RNA. Oh, and there are, as of when this PDF went out, 48 candidate vaccines in preclinical evaluations. So not in people yet, this means in animal models and sometimes it means just in vitro at the experimental bench in cells Okay so DNA-based vaccines, I’m only giving you some of these 48. And then there’s an inactivated virus. So, one of the flu vaccines is just inactivated flu viruses. Like you kill them so they can’t infect you. There’s another live attenuated virus vaccine – that’s the idea of using cowpox instead of smallpox Then there’s subunit vaccines – that means you produce, for example the S-protein, the SARS coronavirus, the SARS CoV2 protein and you inject that in into a person. And, like I said, that’s being tried for HIV and also for influenza Actually there’s a licensed influenza vaccine which is a so-called subunit vaccine. But, like I said, it takes a while to produce large amounts of protein that are what’s called “under good manufacturing protocol” GMP-type of production – so it doesn’t have other things in it which could be very dangerous for you. Okay and then there

are other types of viral vectors where you put the SARS CoV-2 into the backbone of a different type of virus. And I’m giving you examples here that I picked out. Some of these have been tried for other viruses, and you can see there who’s doing them. And you should just keep your eyes out, this is a pox vector like I was talking about. Okay, so I want to close by saying a few things One of them is, this has been an amazing time, even though it’s so awful for the world and for people who are getting sick and infected. I have to say that it’s been an interesting and in some ways good time for science because I’ve noticed a lot more collaboration with a lot of people in many different fields now coming together to try and work on this pandemic. So, I want to thank them and I’d also like to thank people in my lab who’ve now switched to SARS CoV-2 to projects which are allowed to proceed at Caltech in a limited capacity if they’re related to Covid-19. This is a subset of my lab that are working on this, and I really want to thank them for for working on it, and to thank the rest of my lab who are giving us ideas, attending lab meetings virtually I wish I had time to talk about all of them, and what they’re doing. I’d also like to say I’m sorry if I didn’t put all the references in, I wasn’t able to cite all the possible work that people are doing, both around the world and in my lab. I’m sorry I’m not going to name all the people but I wanted to thank my lab for doing science remotely thank the Covid-19 researchers in my lab This is a recent picture. These are people in my lab and also people in the protein expression or another Caltech facility that is the only reason why we can make all these different proteins for vaccine design and antibody research. And this is used by multiple labs across the campus Okay and then I wanted to point out Christopher Barnes, who’s heading up the Covid-19 research in my lab, and also Anthony West. They fielded questions about this and are helping me with answering a lot of these questions I wanted to give you some references for some of the things I talked about. There are multiple other references, these are references in case you’re interested in the candidate vaccines and also a compilation of drug trials and vaccines Okay, thank you very much for your attention