10. Biomolecular Engineering - Engineering of immunity - Cont.

Professor Mark Saltzman: Great, well welcome, today we're going to continue talking about vaccines. We started on this topic on Tuesday, particularly emphasizing smallpox and kind of the history of vaccine development. Then, also emphasizing in the case of smallpox, how even after the scientific discovery was made, it took many, many decades for people to be able to produce the vaccine in large enough quantities and distribute it, so that you could think about making an impact on global prevalence of the disease. We want to talk about that same concept today in terms of polio, which is a vaccine that is both made and manufactured in a different way than the smallpox vaccine. That will lead us into a discussion of sort of modern methods and sort of the spectrum of methods that are available now for vaccine development. The other thing that I want to do today is try to tie this discussion on vaccines a little bit more closely with what we talked about last week, in terms of what happens inside your body when you receive a vaccine or when you're exposed to an antigen, and how the immune system actually responds to that. The question is 'what happens after a vaccine is introduced into the body?' I want to spend some time on that until we talk about--before we talk about development of the polio vaccine. Here, I've just picked a couple of the pictures that I showed you last time when we were talking about cell communication in the immune system, What happens after the vaccine is introduced into your body is that it initiates cellular events. Cells share signals with each other, and that leads to activation of a specific cell population if we're thinking about a vaccine that produces antibodies, for example, that leads to activation of B-cells, immature B-cells, which do two things. They both proliferate, increase in number, and they differentiate; they differentiate from immature B-cells into antibody producing cells. So, let me go back to what we talked about last week and illustrate that a little bit more closely. One of the things that happens is that certain cells within your body process the vaccine or the antigen and we talked about that. We talked about host cells that are perhaps infected with a virus, displaying pieces of that virus, antigenic pieces of that virus in the context of a surface receptor called MHC-1, presenting that. Other cells in the immune system recognizing that this is a foreign molecule, but is being presented in the context of a 'self' cell. Because it has MHC-1, your MHC-1 on it, this T-cell recognizes that it's one of your cells but it has a foreign antigen associated with it. In this case, it might be a piece of a virus that's replicating inside this cell. So, that's antigen presentation to this population of cells called cytotoxic T-cells, T_c, a subset of the class of T-cells in the immune system. They become activated and they produce, eventually, mature cytotoxic T-cells in large numbers. These cells can now kill cells that have the correct signature, and the signature is MHC-1 with this foreign antigen associated with it. Now, for antibody production it is still a T-cell that recognizes the antigen presenting cell. But this antigen presenting cell is more likely a professional antigen presenting cell, or a subset of cells of your immune system that are specialized in ingesting foreign particles and displaying their contents to the rest of the immune system. So, the classes of T-cells: macrophages, natural killer cells, these are a class of cells that's particularly important called dendritic cells. They might ingest extracellular antigen, presented pieces of it on their cell surface in the context of MHC-2. Another subset of T-cells called T helper cells will recognize that signal by direct contact with it, and they will become activated and proliferate. Now, these T_h-cells, helper T-cells, go on to stimulate B-cells, and it's these B-cells that become the mature antibody producing cells that make quantities of antibody that fill up in your body. The antibody that they stimulate is antibody that's specific to this antigen that was presented earlier. Now, I recognize--well you should recognize that this is a very simplified view of a highly complex network of interactions that takes place. If you go on to study more about immunology, which I know most of you will, you will recognize that I'm--this is just the simplest level of one of the most complex systems within our body. It has to be complex, because we're asking the immune system to be able to respond to every potential foreign pathogen that we come into contact with. It does that through a complex set of sort of cellular interactions, and it turns out also gene rearrangements if you go further to study that. This is just a highly simplified view. What I want you to remember is that specific sub-populations of cells get activated, the activation results in a specific response. In this case here, you're generating host cells, cytotoxic T-cells, that can kill only very specific cells, cells that are expressing this foreign antigen. In the case of the helper cells they stimulate a specific population of B-cells to mature into antibody producing cells, and that antibody is generated against the antigen that stimulated it. If we just thought about that second part of it, just the antibody generation, or the humoral what we called last time--last week, the humoral immune response, the immune response associated with generation of antibodies in the blood and in other fluids. We looked at the kinetics of this response, what happens in your body after you're exposed to an antigen. So, this is a time course here, this scale is in days. So, this is several months and this logarithmic scale on the Y-axis represents antibody concentration. Now, we're not thinking about total antibody concentration because you already have a lot of antibodies circulating within your blood and in your fluids. We're thinking about the particular antibody that binds to this antigen that you're exposed to. I'm using, here, antigen interchangeably with vaccine for our purposes today. So, the antigen we're thinking about is a vaccine particularly designed to elicit immune response against a pathogen. You introduce that antigen into a person, into me for example. There's a lag period where if I was just looking for antibodies nothing happens for a while. At some point, maybe a week later, four to eight days later, you would start to see antibody levels rise. Again, these are antibodies that are specific to that antigen or vaccine that we introduced. Those levels would reach a plateau after some period of time, maybe after a couple of weeks and then they would begin to decline again. Now, if I was a person that was designing a vaccine and I noticed that this was the response that it got, that antibodies were produced, they reached some intermediate level, they started to fall, I would say, 'well I haven't stimulated the immune system enough, let me re-boost, let me give another dose of antigen. ' If you did that what would happen is you would see antibody levels rise even more sharply than before. The response to the second exposure in antigen is different in a couple of ways. One is there's no lag period, notice that antibody levels start rising right away after the second exposure. That rate of rise is steeper so they--antibody levels go up more rapidly and they reach a higher level. Now, this is just a typical response. You could probably find some antigens that don't follow exactly this behavior, but in general, this is the kind of behavior you would see on first exposure to an antigen or vaccine, called the primary exposure, and on subsequent exposure to an antigen or vaccine called the boost. If this was tetanus, you got this tetanus vaccine when you were young; you get a boost every five or ten years because your antibody levels are starting to fall. Now, just--what this diagram also shows you is that that response is specific to that particular antigen. It's not just that your whole immune system gets revved up and it's going to respond more rapidly to any antigen it's exposed to. If we did the experiment where on this booster we included not only the initial antigen but some unrelated antigen, the response to the unrelated antigen called B here, looks like a primary response. There's a lag phase, there's a slow rise to an intermediate level of antibody. So, every time you're exposed to a new vaccine or a new antigen you go through this primary response before you have the secondary response. Does that make sense? Kate did you have a question?Student: If there were just--if you were trying to create a positive reaction of antigens and it showed up naturally wouldn't it create this reaction anyway in terms of your body would create antibodies like the secondary response volume to antibodies?Professor Mark Saltzman: So, I'm not sure I understand the question. The question is, 'if you're naturally exposed to antigen wouldn't this happen anyway?' Yeah, so for example, you get exposed to--before there was a vaccine your brother or sister had chickenpox, and so you got exposed to chickenpox naturally through your contact with them. You would have this initial response, now that initial response might be too slow to prevent you from getting chickenpox.Student: Right, but if you just got the primary exposure, wouldn't the secondary response automatically--not a booster shot, just the secondary response [inaudible], because you were exposed [inaudible]?Professor Mark Saltzman: Yeah, so you're asking, if for example, why do you need a booster of tetanus, because if I get exposed to tetanus wouldn't I have this rapid response? The answer is 'yes you would', the question is 'would that response, even though it's much faster, be fast enough to protect you from the initial exposure to tetanus that you got?' Probably it wouldn't, you would probably get a little bit sick anyway, but recover. That's a really good question, and I'm talking in terms of generalities here but the specifics matter. That's why every--development of every specific vaccine turns out to be different because they don't all follow exactly this kind of time course. Some, like the smallpox vaccine on one exposure generates a very high response that lasts for many years so you don't need a boost. Others generate a weaker response that does require boosting. So, there's no absolutes about this, this is a general response where all the features can be different with different pathogens. Did that answer your question? Why the lag phase? Why the lag phase at the beginning? Well, because it takes some time for these cellular events that I mentioned earlier to happen. The antigen has to be presented to helper T-cells, those helper T-cells have to stimulate a B-cell population to both proliferate and differentiate. So, this is a picture I showed you before. You can imagine that even when this immature B-cell gets the signal 'now is the time, you need to turn on antibody production', that it takes some time for it to both proliferate to make enough cells and for those cells to mature to the point where they become what are called plasma cells, which are antibody producing factories; takes some time for that to happen. Now, why is the time less on second exposure? Because on second exposure there's another population of cells that I haven't mentioned before that remain after the primary exposure and those are called memory cells, they're down here. So not all of the B-cells that are stimulated become plasma cells or antibody secreting cells. Some of them become what are called memory cells. These are cells that recognize a particular antigen, they're ready to differentiate into antibody. They're ready to rapidly differentiate into antibody producing cells and they're waiting for that second signal to come. So, these memory cells are a way that your immune system keeps track of antigens that it's been exposed to for even if maybe the plasma cells that were producing antibody in response to the initial exposure have died and disappeared. Memory cells are long lasting cells that remember this exposure and can respond very quickly on second exposure. We talked about antibodies, we talked about them two weeks ago, we talked about them in section last week, uses of them. I just want to remind you that if you looked at the population of antibodies inside--in your blood, for example, the predominant antibodies would look like this. These are of the class called IgG, they're Y shaped molecules. They have a region down here called the FC region, and that is responsible for effector functions. There's a region up here called the antigen binding region and those--and there's two copies of that region and it's responsible for antigen binding. So, many--the predominant number of antibodies in your blood look like these IgG molecules. But not all of them do, there are different kinds of antibody molecules. Not only the IgG but there are special antibodies called secretory IgA and these are highly enriched in mucosal fluids in the mucus lining of your gut, and the eye, and of other--of mucosal organs. They're also enriched in milk. So, milk contains large quantities of this special class of antibodies called secretory IgA. They also have binding sites for antigen, but they are sort of two IgG type molecules bound together by another peptide chain. So, imagine taking two IgG's, turning one upside-down and then they're hooked together. The advantage of this is that now you have four binding sites for antigen instead of just two. So, these are better at binding to antigen because they have more binding sites on them. It also turns out that they're made stable in these environments like milk and mucus secretions because of this secretory chain which is wrapped around it. Another important class of antibodies is called IgM. The IgM is really five IgG-type molecules that are linked together through disulfide bonds, such that their FC portions are all pointing in and their antibody binding portions are all pointing out. So, now you have a single molecule, very large molecule, with not just two binding sites but with ten binding sites. This is a very potent molecule for binding to antigen. One of the things that I didn't mention before is that when you get this primary response and then the secondary response, if you looked at the antibodies that are generated during the primary response, again we're only looking at antibodies that bind to the particular antigen or vaccine that we have used for the priming. If you looked at the antibodies that were present in the blood, for example, you would find that most of those antibodies are IgM during this initial period of antibody concentration rise. Most of them are of the class IgM; IgM antibodies are produced on first exposure. If you looked later, as the antibody production response matures, some IgG is produced so that in the late period after initial priming you'd have a mixture of IgM and IgG in the blood. On second exposure it's different, that IgG is produced predominantly on second exposure to an antigen. One thing I do want you to remember is that IgM class antibodies are the antibodies produced on first exposure. Why? Why do you think IgM are produced on first exposure? Well, one way to think about is they have more antigen binding sites and so they're going to be more efficient at neutralizing the pathogen on a per-molecule basis than IgG is. So, it's good to get those produced more quickly. The memory cells, which are stimulated, lead to an IgG response and that's why IgG is the antibody of--that is produced predominantly after the boost, but there is some IgM produced also. Let's talk about the polio virus vaccine, keeping those things in mind. Polio was--is a crippling disease. In many cases, it affects--it also initiates its infection through the gut. It can be passed from one person to another orally and infects first cells of your intestinal system and then spreads to other cells, in particular, spreads to cells that are involved in the neuro muscular junction and can affect then muscle activity or your ability to move voluntary muscles. So, polio--the disease caused by polio can be a paralytic disease, crippling, and in some cases can lead to death if the disease progresses in certain ways. If we looked in 1950, this is the incidence of paralytic, or the worst form of poliomyelitis in the U.S. was about 20 per 100,000 people. This is mostly a disease that would occur in children. You would first get exposed in children--in childhood and then at a point when you're susceptible to the disease. So in a town that's the size of New Haven with a population of let's say 100, 000 people in just the immediate New Haven area, there might be 20 of these instances of very severe form of polio per year, 20 crippled children would result. So, over the course of time this could have a very substantial impact on the community. Because it's passed by--can be passed by an oral route it's a disease that's very effectively transmitted in school settings where children are together, or childcare settings. So, it was something that parents before 1950 were very concerned about. If a case of polio emerged in the community, the chances that it could spread to other children or to your child were high; so, great interest in this in the early part of this century. A group of scientists, mainly in Boston found, importantly, that they could cultivate the polio virus, the disease causing polio virus; they could cultivate it in cell culture. They found that certain cells, in particular, epithelial cells from monkey kidneys, were very effective at propagating the virus. So, you would grow these monkey kidney cells in culture, you would add some virus to the culture broth, the cells would become infected, the virus would go through its life cycle. The cells would be basically little reactors for generating lots of virus so you could make lots of virus to study. Jonas Salk, who probably you've heard the name, was a physician who, at the time thought, 'well if we can make large quantities of this virus then perhaps we can make it into a vaccine.' But unlike the Cowpox virus, vaccinea, that we talked about before, this is the real disease causing agent. If you just introduced this polio virus, which you could make in large quantities into people now, you would be causing polio. So, you couldn't introduce the live virus in because that would cause the disease not just immunity. Remember that the lucky thing about the smallpox vaccine was that a naturally occurring attenuated form of the smallpox virus variola called vaccinea was found. So, that was a naturally occurring attenuated virus that could be produced into a vaccine that didn't cause the disease. The strategy that Salk used was to kill the virus instead. Make a lot of the virus, it has all of its antigenic epitopes on it, but we'll just kill it so that it can't replicate. Then, if we inject it into people they'll be getting the real virus. Hopefully their immune systems will respond to it like the real virus but it won't be capable of replication because we've chemically cross linked it so it can't go through its life cycle. I'll talk about viral life cycles in a moment and you'll see how that killing worked. They grew the virus in monkey cell cultures, they purified the virus because you got to get all the other stuff from the cells that you're growing it in a way, they inactivated it by treating it with formalin which is just a formula--it's just a mixture of Formaldehyde; Formaldehyde cross links proteins. So, you cross link all the proteins in the virus, and you make a particle that looks like a virus but it can't act like a virus any longer because it can't replicate. Then, they did preliminary studies of safety and effectiveness in people. Basically, injecting it into some test subjects, making sure that they didn't get diseased from it and looking at antibody responses to see if it worked and it did. So, very rapidly a clinical trial was started. Now, the problem, or one of the challenges with clinical trials of vaccines is that you have to enroll a lot of patients into clinical trials because only a few are going to get sick in any case, only 20 out of 100,000. So, you're treating healthy people and you're trying to prevent them from getting a disease and you don't know who's going to get it. You have to test it by giving the vaccine to a large population of people, and then watching and seeing if you've reduced the incidence of the disease. It's--that's a very different process than testing a drug, where if you had a drug for heart disease, for example, you would give it to patients that had heart disease and see if you had an impact. You could do that with a relatively small number. If you're trying to prevent a disease that only occurs at a rate of 20 per 100, 000 people you have to give the vaccine to millions in order to see if the number goes down. Does that make sense? They started the clinical trial. The clinical trial was designed such that almost two million elementary school children were given this test vaccine. You could imagine that this is a monumental sort of undertaking in a number of different ways. One is if you have to coordinate how you're going to give this vaccine to two million children across the U.S. You want to give it to people in different communities, to make sure that it works in all the subpopulations where the vaccine's potentially valuable. You want to give it to children because it's children that are susceptible, and that's where you would like the vaccine to be useful is in children. So, you want to give it to them because the biology of children is different than adults, and so you need to make sure it works in that population. They had to give some of them the real vaccine and some of them a placebo vaccine in order that they could really tell if the vaccine worked; you have to have it placebo controlled. They did this in 1,800 elementary school children, so these are about eight year-olds. So, imagine proposing a clinical trial like that today where you had a test vaccine that had been tested in a few patients, was thought to be safe, but we're going to give it to a million--the test vaccine to a million or two million eight year-olds in the U.S. and see if it works. Well, you know that was possible at this time for a couple of reasons. One is people must have had an incredible amount of confidence in Jonas Salk. He did a good job in preparing the initial studies to show that it was safe. Two is it gives you some sense for how concerned parents were about the risks of polio in the community and how much they wanted a vaccine to be developed, such that they gave permission for their children to enter into this trial. Well, the vaccine turned out to be about 70% effective. As we'll see in section today, a vaccine does not have to be totally perfect in order to prevent transmission of a disease, because when a disease enters a community its spread from one person to another. If you can block one of those people from getting the vaccine, you also stop--from getting the disease--you also stop all the people they would have transmitted it too from getting a disease. One can stop spread of disease through a community without being 100% effective in each person who gets the vaccine. That's an important concept. So, it was effective, it was rapidly then introduced into general use. I taught at Cornell before Yale, and my assistant was a woman named Bonnie at Cornell. She was part of the clinical trial that did this, and they gave everybody certificates after it was done. So, you didn't know at the beginning, you knew you were enrolled in the trial, they gave you a shot, you didn't know whether you were part of the real group that got the test vaccine or the placebo group that got the control; turned out that Bonnie was part of the control group. So she got a certificate at the end thanking her for participating in the clinical trial, and also telling her to go get the real vaccine because she hadn't had it yet. So, this is real people who were involved in these tests. Well, this shows what happened in the period after this vaccine was introduced into the general population so that would have been in 1954. This is a complex slide, so let me show you what it is. The Salk Vaccine is also called the Killed Polio Vaccine, and some people call it KPV, also sometimes called Inactivated Polio Vaccine, IPV, but this is the vaccine I just talked about produced by Salk. After the clinical trial it was rapidly introduced into the population. This curve here with the square shows you how many millions of vaccine doses were distributed across the U.S., so this is hundreds of millions of doses that were given. As those doses were given you look at the prevalence of polio within the United States. It dropped dramatically in the period from 1954--this is these filled black bars refer to this axis, polio cases per 100,000 population dropped down to only three or four cases by 1956. So, this just shows as the vaccine was distributed, given to more people, that prevalence of the disease dropped dramatically. Well, it also illustrates that one of the things you do after you introduce a vaccine, you can't stop there, you have to continually watch what's happening with this disease in your population. One thing that happened was that after 1956,1957 the number of cases were down, there was a small bump here, the cases were up. This was of great concern because the number of polio cases shouldn't go up as the vaccine is being even more actively distributed through the country, so what happened? This led people to go back and look at the places that were manufacturing the vaccine to make sure that they were all producing vaccine of the proper quality. It turned out that one of--there were three vaccine manufacturers, one of them was using the procedure not quite correctly, they weren't completely killing the virus when they produced their vaccine. So, some of these cases were probably due to polio that was transmitted by incompletely killed virus that was present in the vaccine. They fixed that procedure and after that the cases went down even more. Now, the polio vaccine that Salk produced was very effective, but it required a fairly large dose of the vaccine and it had to be injected into the arms of children. So, there was some thought that maybe we could do better. Particularly, if we took advantage of the fact that this is a virus that's easily transmitted orally and could you make a vaccine that would be effective orally as well, that would be a tremendous advantage, especially in children who don't like to get shots. If you could take your five year-old or eight year-old in to get a vaccine that was orally administered instead of a shot that's a much easier thing to do. Plus it makes it, as I talked about last time, much easier to think about distributing the vaccine around the world because shots require skilled medical personnel, whereas, an oral vaccine could be self-administered. That means that it's easier to take into certain kinds of populations or remote parts of the world. An oral polio vaccine was developed by a man named Sabin. What he did was took the polio virus into the laboratory and tried to make an attenuated form of it, that is, 'can I get the virus to mutate in ways that it doesn't change its physical structure much so it still looks like active polio but it changes its disease causing properties, so it changes disease causing properties without making it non-immunogenic?' He produced an oral polio vaccine from an attenuated virus. This was not a naturally occurring attenuated virus as used in smallpox, but a virus that was attenuated in the laboratory, basically by propagating it in culture and looking for mutants that were formed as you propagated this virus under different experimental conditions. This is the vaccine that you probably took; the vaccine that's still most widely used in the U.S. is the oral form of the vaccine. It's used because of the reasons I described. Why would you maybe not want to use the oral vaccine? What are the disadvantages of using it? Knowing what you know now, any concerns about taking the oral polio vaccine instead of getting the shot? Are there any features that you'd worry about? Bobby?Student: The virus is not killed so [inaudible]Professor Mark Saltzman: It's a live virus, which is actually going to infect your intestinal system and reproduce. Because it infects your cells and reproduces, your immune system responds much more vigorously. You could imagine that you've got virus that's propagating inside your cells, making more and more virus, your immune system really responds well to that. Doesn't respond as well to killed vaccines, and that's why the Salk vaccine has to be injected at a high dose. So, it's more effective because it's a live virus but it's a little bit more concerning because it's a live virus as well, in that you trust that it's attenuated but could it convert back to a virulent form or a form that caused a disease. Turns out that that hasn't been a problem. In fact, another advantage of the oral vaccine is that you give it to children. They take it, the vaccine itself, the virus, reproduces in their gut and they can actually spread it to other children in the same way that they spread the disease where you've got children that are maybe at school or at childcare. Have you ever looked at children in the playground? They're all over each other sometimes and they can spread saliva or other fluids. It turns out that if you give one child in a home the oral vaccine, you often have a protective effect in other children in the home as well because it spreads from one individual to another. That's another advantage of the oral polio vaccine. Well, polio is not yet eradicated but there still are hopes that polio could be eradicated. It's only endemic, that means only naturally occurring in certain countries. The World Health Organization keeps track of what countries have cases of polio and when they occur, and what the frequency of--So, this is a map from a few years ago and there are efforts that occur occasionally. For example, this effort that's described here from 2001 where the World Health Organization has a push, they say, 'we know where the cases are occurring, we know what communities still have polio within them, if we do sort of really gear up for a massive immunization effort in those areas we could eliminate polio from that community. In this way, by knowing what communities it's in and acting on all of them at once you might be able to eradicate polio in the same way that we eradicated smallpox.' So far those efforts have failed for a variety of reasons. One is the resources that are needed in order to do this. The other is that some places where the disease occurs the governments are not stable, or there might be civil unrest or civil wars and that makes it very difficult to orchestrate giving vaccines when there's other things happening in the country that are of more immediate concern. And there are some communities that are frankly suspicious of Western medicine and don't want people to come in with their modern approaches and feed things to members of their community. So, there is still problems to solve in doing this. I wanted to show you, so that if you're interested in this, and you want to keep track there is a website called Global Polio Eradication Initiative and you can look, and you can actually look and see what countries polio is occurring in and where they are, and how many cases have been reported. You can't see this too well but there's a map of the world here that actually shows you all the individual cases of polio that occurred between this period of August 2007 and, it's cut off on the screen, February of 2008. This is the kind of surveillance that's needed to really make an impact and this is why--one of the other ways where engineering approaches are needed in order to solve medical problems like this one. There's a lot of engineering that we've already talked about in terms of producing quantities of the vaccine, producing it reliably, producing it safe, distributing it, and keeping track requires a level of sophistication that maybe you wouldn't have thought about initially. That slide is on your--is in the slides that are distributed if you want to follow on that website, there's the picture that I copied yesterday. You see most of the cases are in Central Africa and in the region around India, particularly Northern India. Let me go back and finish up today by talking about the lifecycle of a virus. Again, this is a highly simplified version of the lifecycle of a virus. This might be a polio virus, for example. The example I've given here is a virus that contains DNA as its genetic material. You know that some viruses use RNA as their genetic material and so their lifecycle is going to be slightly different than this. *** is a member of the family of viruses called retroviruses, and retroviruses all use RNA as their genetic material. I'll talk about that at the end of the lecture here. Here's a DNA virus, it infects a cell. Usually viruses have certain kinds of cells they want to infect or that they're capable of infecting. That infection occurs because of a ligand receptor interaction on the cell surface where the virus itself is the ligand and it takes advantage of a receptor that's expressed on the cell surface. For example, *** enters cells of the immune system by binding to a receptor called CD4. These tropisms, or affinities of viruses for certain cells, are well mapped out now. The virus enters the cell and it breaks down. It breaks down into its component parts and I show two of those component parts here, one is the genetic material, in this case DNA, and the other is all the proteins that form the structure of the virus. That DNA gets replicated to make many more copies of the viral DNA using host mechanisms, that is, often using the DNA polymerase which is naturally present in the host cell for its own replication. The proteins that are produced by genes that are on the viral genome get transcribed and translated in order to make more structural proteins that are needed for assembly of the virus. The virus then can self-assemble, that is, you've made a lot of genetic material, you've made a lot of the structural pieces, there has to be some way that the virus can reassemble, repackage itself into active forms. Then that--those active forms are released from the cell. Now, sometimes those--that release occurs without the cell itself dying. In other cases, the virus propagates in such high numbers that release is literally an explosion of the cell. Release of tens of thousands, hundreds of thousands of new virus particles from one individual cell such that the cell gets killed in the process of replicating the virus. These released particles can now go on and infect neighboring cells, they can travel in the bloodstream to infect cells at a distance and the virus spreads throughout a multicellular host. Now, what happened with the Salk vaccine is that Formaldehyde was used. Formaldehyde cross-links proteins, so, if you treated this virus with Formaldehyde, even it was able to enter a cell, it couldn't break down anymore. So, its genetic material wouldn't be released and even if it was released, the genetic material is also cross-linked, and so it can't be transcribed and translated or replicated. So, this is the stage at which you prevent disease in the Salk vaccine. In the Sabin vaccine, or the oral polio vaccine, now you have a non-virulent virus. So, one that perhaps does not reproduce in such high numbers that you create an overwhelming infection, but one that still goes through its lifecycle but is limited in its effect. That suggests, now, if you think about this even highly simplified lifecycle, suggests some other ways that we might use kind of modern technologies to engineer new vaccines. We've talked about getting lucky, finding a naturally occurring attenuated live vaccine as in the case of smallpox. We've talked about killing a virus by cross-linking it, for example, to make a substance that looks like a vaccine--looks like a virus but can't replicate. We talked about attenuating in the laboratory, using cell culture techniques and what we know about mutating viruses. One can also purify parts of the protein, that is, parts of the virus, that is, 'do I really need to deliver the whole virus?' If the immune system recognizes only small pieces of the virus and mounts an immune response to that, how about if I just take these pieces of a virus like some structural subunit, some piece of protein and use that as a vaccine? Now, I'm not introducing any genetic material at all so I don't have to worry about it replicating because there's no genetic material, all I do is deliver the particles. Well, this is an approach that's been used in a variety of vaccines, most successfully with Hepatitis B, so the problem is where do you get these proteins? Well, one way to get proteins, and it was first used in Hepatitis B, is to find patients, find individuals who are already infected with Hepatitis B. So, Hepatitis is already infecting cells of their liver, their liver is actively making new virus. It turns out in the case of Hepatitis B, the way the lifecycle proceeds--the cells make too much of the protein and not all of it gets assembled into the virus. So, if you look in the blood of patients that are infected with Hepatitis B you find a lot of Hepatitis B surface subunits, proteins without the nucleic acid circulating in their blood. What if I collect that blood from patients that are already infected with Hepatitis B, purify the Hepatitis B protein, and inject that back into people? That would be a subunit vaccine because I'm purifying a subunit of the virus that could be injected and hopefully induce an immune response. It turns out that that works. Any potential problems with that approach? Would you like to get that vaccine? You've all been immunized for Hepatitis B, would you be happy to hear that that's where your Hepatitis B vaccine came from? It's okay? Sounds okay? Student: [inaudible]Professor Mark Saltzman: It does sound okay and it does work. The danger is that it's being purified from patients that have a disease and so you want to make sure that there's not other diseases that are present in that sample at the same time. The Hepatitis B subunit vaccine was produced at the same time that ***-AIDS was being recognized as a problem in this country. We did not yet have good methods for screening blood to look for ***, we didn't have the ELISA technique that I--we talked about in section a few weeks ago and so there was a great concern that there might be other diseases that you'd be passing on from--unknowingly from this subset of patients that you're isolating the vaccine from. So, that particular sub unit vaccine was only used in people that are at high risk for acquiring Hepatitis B, that is, people that work in healthcare situations that are exposed to blood routinely as part of their job. It wasn't used in the general population. What was--the vaccine you got was produced totally outside of people using recombinant DNA technology. In that case, we took the gene for the Hepatitis B protein. Took it out of the virus completely, cloned it into a plasmid, that plasmid was expressed in a foreign host, in this case it was expressed in yeast cells. Yeast cells were grown in large numbers with this plasmid inside, they expressed the plasmid and so you made Hepatitis B surface antigen not in people but in cell culture where it was not normally formed. Then that subunit was purified and formulated into the vaccine, the kinds of vaccines that you and I got. This was an early example of recombinant DNA technology being translated into a clinical product and that's the vaccine that's widely used in practice now. Another approach which is still investigational, in that it's being tried for many diseases but not yet clinically used in any particular one, is maybe you don't need to introduce a whole virus or pieces of a protein at all. What if you took, instead of using--instead of going through the step of isolating the gene for Hepatitis B, cloning that into a plasmid, expressing the plasmid in cells, manufacturing these cells, and then purifying the protein product--what if you just took that plasmid that contains the gene for Hepatitis B and you gave that directly to people? Then, you could get the Hepatitis B protein expressed in your cells. If we injected it into a muscle let's say, and your muscle cells took up this plasmid. Now the plasmid started to do its thing, which is replicate and the gene gets transcribed. Then, your muscle cells would start producing Hepatitis B surface antigen and your immune system recognizing that's a foreign protein would start responding to it. That concept is called DNA-based vaccine, or DNA vaccines. So, totally avoids the manufacturing processes that are used to produce other vaccines; you got to manufacture DNA instead. I want to say a little bit about the cost of vaccines because this is a part of what makes it difficult to accomplish what's usually our goal in a vaccine development, which is deliver the vaccine to every population in the world. Often, vaccines cost a considerable amount to produce. The Hepatitis B vaccine I talked about before produced by recombinant DNA technologies, called Recombivax HB, that's one version of it. If you or I buy it, it costs $51 per dose, you need three doses to be effective. Why do you need three doses of Hepatitis B? Because this is not a virus at all, but it's a virus subunit, your body doesn't respond to it as strongly, your immune system doesn't respond to it as strongly. So, you have to formulate it properly. That is, mix it with things which make it more--make your immune system respond more strongly. You have to inject it multiple times because the first blip that you get in immune response is not very high, you have to boost and often you have to boost again in order to get a high enough response to be protected. For any one of us it costs $153 - $156 to be immunized. Now, that is doable for us but that's not affordable in many parts of the world. In addition, this is just the cost of the vaccine, not the cost of the doctor or nurse who injects it into, you so you have to figure that cost in as well. The CDC can buy this from the manufacturer for a lower price, and when you hear about government organizations distributing vaccines to different parts of the world, they're buying at a reduced rate but it's still not inexpensive. Measles, Mumps, and Rubella this is an established vaccine also quite expensive. I didn't have the commercial price for the chickenpox vaccine called Varivax but you can imagine that it's even more than $50 a dose for that one. I just wanted to try to put that in perspective. I also talked last time about smallpox and the perceived need to produce more smallpox vaccine in the event that smallpox is used as a weapon in 2002. So shortly after 9/11 the Government made a contract to a company called Acambis to make four hundred million doses of smallpox for $343 million dollars. So, this is not cheap, right? The problem is you've got to make hundreds of millions of doses sometimes in order to have an effect on progress of the disease. So, even if the cost is small, $10 a dose, it quickly amounts to a large amount of money. There were some problems with that deal and I just give you one news report on that, but you can follow it if you're interested. In spite of that fact, it turns out that vaccines are one of the best uses of our money in terms of extending the lives of population. This is old data now, from 1995, but I don't think it's changed very much. It asks the question, 'how much do different public health interventions cost per life saved?' So, we have a mandatory seatbelt law here. That means that you have to have seatbelts in all your cars; that means people pay more for cars because they have seatbelts. You have to enforce the law and all the costs that goes along with that. In terms of lives saved by that measure, it's estimated that it cost about $69 per life saved, so that's a reasonable cost to spend. For something like Measles, Mumps, and Rubella immunization which costs what I showed you before, you can save so many lives that way that the cost of distributing and producing the vaccine is actually less than the value of the lives that are saved. So, it saves money, you're saving money by doing it, not that it's not costing you. Obviously, these are complex calculations, but I just want to point out that and smoking cessation advice, advice about not smoking to pregnant women is another very inexpensive life saving intervention. Things that we think are good, and I'm not advocating we don't do them, like having radiation emission standards for power plants, nuclear power plants, and other power plants cost a lot of money per the risk involved with them. On this scale, vaccines are a very inexpensive way to save lives. Okay, we'll stop there, section this afternoon, we'll talk about disease spread through populations and how vaccines impact that.