How we Deal with Virus Infections
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Lindau Nobel Laureate Meetings237 / 340
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00:12
Grethen Sonnier, Liebekelege Studente. I think that exhausts my supply of German and the rest of this will be in something
00:20
approximating English. I'm going to talk about infections, particularly virus infections. I didn't expect to also be giving you a practical demonstration, but at the moment I have the flu or something like it, which just goes to show we haven't been very good at dealing with virus infections. There are many that we haven't dealt with.
00:40
Now, the word immunology comes from the Latin term immunus, which means without tax. That referred to the fact that certain members of the Roman state, former soldiers, were exempt from tax. The tax that the immune system has evolved to remove is the tax that's caused by infection. This has been the selective force that has driven the evolution of the immune system.
01:05
The idea of being without tax, of course, has considerable appeal to many conservatives, particularly in the United States, but there is no life without tax, as you will see from my performance at the moment. Now, by the late 1960s, many people were thinking that the era of infectious disease
01:23
was essentially over. This is one of my scientific heroes, Frank Macfarlane Burnett. Mac Burnett won the Nobel Prize for immunology in 1960 with Peter Medawar for studies of immunological tolerance. In actual fact, Burnett was a virologist by training. He did some of the seminal experiments of virology.
01:42
He was the first person to do quantitative virology, and of course, if you don't do quantitation, you don't do anything very much, firstly with bacteriophage and then with mammalian viruses. And then, somewhat late in his career, he focused his attention mainly on the immune system. And by the late 1960s, when he wrote his autobiography in 1968, he was convinced that
02:05
intellectually, at least, the era of infectious disease was over. Now, as we now know, that is not true. He was wrong, that we have enormous intellectual and practical challenges in dealing with infectious disease, the AIDS epidemic, even simple respiratory infections like I
02:22
have at the moment. We have not dealt with them terribly well. I bring Burnett into this because he is one of my personal scientific heroes who had some influence on me. He spoke three times at Lindau, I see, from consulting the book, and he would have been 100 years old if he had lived. This is his centenary, and in Australia, we're celebrating his centenary next month,
02:47
in fact, in Melbourne. Now, the reason that Burnett thought that the era of infectious disease was largely over was that we had, at that stage, had enormous success with vaccines. This is what happened when polio vaccine was introduced, the Salk vaccine.
03:05
You can see the incidence of cases here. It was a terrifying disease. Many people were enormously frightened of it, and there were large numbers of cases. And you can see that the vaccine pretty much knocked that infection right out. There were some early mistakes and so forth, but we are now almost at the point
03:22
of eliminating poliomyelitis virus from the world. The Children's Vaccine Initiative has demonstrated, has delivered, enormous numbers of poliomyelitis vaccine doses throughout the world, something like 10 million doses alone were given one day in India in a pulse vaccination program that's hoped will eliminate polio by 2002.
03:45
So all of us are aware that there are some infectious diseases that we can get rid of. There are others that have also been eliminated. Smallpox is gone. That's 200 years after the first report of that vaccine, the first published report
04:00
of that vaccine, or written report by Edward Jenner, the English physician. It doesn't normally take us 200 years to develop vaccines at the moment, fortunately. Polio is on the way out. Many others are very well controlled. Measles, as it's thought, could also be eliminated. There are other viruses and other infections for which we actually have good vaccines,
04:21
but we still have substantial mortality in the world population. Yellow fever virus, for instance, will kill a number of people in Africa every year. We've had a very good vaccine for that, Max Tyler's vaccine, since 1957. But the fact of the matter is that it's not distributed widely because of cost. And this, of course, in the developing world is a major factor, the cost of vaccines.
04:45
And we have to be able to deliver cost-effective vaccines, even if they aren't good vaccines. This is a problem for us, especially as we talk about trying to develop AIDS vaccines. Many of the AIDS vaccines that are currently under discussion are extremely expensive products, and we would have to see how we can actually give those.
05:03
I believe the funding would be there for that if we actually had a product that was successful. But at the moment, none of us, I think, can guarantee that we can make an AIDS vaccine, though there are something like 300 different vaccines in various stages of testing at the moment. There are also many other infections for which we do not have vaccines,
05:23
and it seems extremely difficult to make them. And when Burnett in 1968 was saying that we'd overcome the problem of infectious disease, he was not, I think, thinking about the problem of infectious disease in the developing countries, where particularly the parasitic infections create an enormous toll.
05:41
Malaria, for instance, kills something like one child every 15 seconds, I think it is. Tuberculosis is an enormous problem. We've had a so-called tuberculosis vaccine for many years, but it's not a very good vaccine. We need something much better that we can deliver much more broadly. There are others as well. Of course, other vaccines are under test.
06:03
Vaccines against respiratory syncytial virus in children, for instance, is under test at the moment, and so forth. The world progresses, but I would not like any of you to go away from this with any sense that these problems are solved and there's nothing to do in the future. There are enormous challenges here for young people.
06:20
There is enormous complexity in dealing with these infectious agents. They evolve very quickly, they change very rapidly, and the problem of dealing with them is at the limits of our conceptual ability when we think about the immune system. There are going to have to be new insights and new ways of doing it. So here, I think, is a challenge for you.
06:40
The other problem that we face is though diseases like measles can be readily controlled by vaccination, many people in the developed countries have never seen these infections, and a number of people, quite vocal minorities, are refusing to have their children vaccinated against the common childhood diseases. I ran into this in Australia after the Nobel Prize.
07:01
I had a certain amount of public exposure in Australia, and there was a very vocal minority, critical of vaccines, believing that this is some sort of plot between the medical profession and the drug companies who actually don't particularly like vaccines because there's not that much money in them, and academic medicine. It is a problem and there have been outbreaks that have resulted from the spread of these sorts of attitudes.
07:25
It's been less a problem in the United States because it's mandated that all children be vaccinated before they go to school. The Australian government has now introduced legislation which brought up the vaccination rates, which were down as low as about 55% from completion of the childhood vaccines,
07:43
brought them up to about 90%. In fact, to cover the population, you need to develop a level of herd immunity that comes in at about 80% vaccination level. So as long as you can achieve something like 80% vaccination, you can do quite well. But this rejection of vaccines, I think, is part of a general phenomenon
08:03
that we're seeing in Western culture, and that is the rise of the irrational and the rejection of scientific-based medicine and scientific-based approaches. Rather sad, I think. The immune system is complex, enormously complex, in fact.
08:21
The two basic elements of it are the receptors that are highly specific, very varied, and show enormous diversity. They're expressed by two types of lymphocytes, or white blood cells. The antibody molecules that are expressed on the surface of the B-cell and a secreted product of the B-cell lineage, the plasma cells. These are protein molecules that are secreted into the bloodstream
08:42
and they bind to protein molecules and act very well to deal with isolated proteins. The other receptor that I'll talk about most in this is the T-cell receptor, which is embedded in the surface of effector cells. In this case, the effector is the molecule, the antibody molecule. We produce enormous numbers of these molecules.
09:02
They flood the immune system and they stick around for life. In fact, the B-cells or plasma cells that make these molecules after stimulation, after priming, will stay in the bone marrow for many, many years, certainly for the life of a laboratory mouse. The immune cells that bear these receptors, on the other hand,
09:21
are somewhat more limited in availability because the development of this response depends on the doubling time of those lymphocytes. I'll expand on that as I go through and talk about the nature of cell-mediated immunity. Now, let's talk from now on about virus infections. We'll deal with relatively simple virus infections,
09:42
the negative strand RNA viruses. These are the viruses that commonly cause respiratory tract infection. This is a parainfluenza type 1 virus, which causes croup in small children. They have a nucleic acid core, they have various proteins in them to keep the nucleic acid protected, and they have surface glycoproteins
10:01
which are involved in such functions as getting the virus into the cell or getting the virus out of the cell and so forth. This is the surface glycoproteins are where the virus is vulnerable to immune attack by antibody, and this is where the antibody molecules will bind to these surface molecules. The cell-mediated system, on the other hand, can draw from any protein that the virus makes
10:22
within the cell, and the immune response can be directed against a peptide of any protein from the virus. It doesn't have to be on the surface of the virus. This is how antibodies bind. This is an X-ray crystallographic picture from Peter Coleman, Graham Laver, Rob Webster's work,
10:40
showing the hemagglutinin, one of the surface molecules of the influenza virus, and they worked out by a combination of selecting viruses with monoclonal antibodies and by X-ray crystallography that these are the actual contact sites for the antibody molecule. The point I'm making here is that the antibody molecules bind in the main
11:01
to tertiary structure of the protein. That is, it's not generally possible to make a linear peptide or linear component that will actually give you good protection against a virus because it depends on the proper folding of the molecule that you will get the appropriate binding that will then bind
11:20
the infectious agent itself. Most vaccines are based on that interaction, the antibody-protein interaction. Most of our successful vaccines work by antibody. The most successful vaccines are those that actually deal with infections that have to go through the blood
11:40
to cause disease or pathology. The reason for that is it's possible to keep reasonably good antibody levels in the blood. If you're vaccinated against yellow fever virus, you will still have antibodies that are readily detectable in your blood 10 years later. Now, that means to actually get the disease yellow fever,
12:01
the virus has to get from the mosquito that injects it it actually replicates the mosquito also to the liver where the damage occurs that causes yellow fever. It's yellow fever because the virus destroys the liver and gives you jaundice. And so, once the virus gets into the blood stream, the antibodies can grab it and take it out of the
12:21
circulation by neutralizing it. So those vaccines work very well. Other examples of this are poliomyelitis and measles. Most vaccines, however, do not give you complete sterilizing immunity. The poliovirus, for instance, still gets in, it grows still in an immune individual
12:41
it can still grow in some of the superficial epithelial cells of the mucosa and then, but cause disease, it must go through the blood and get to the large spinal motor neurons which are damaged by the poliovirus and cause the disease we know as poliomyelitis. Now, at the worst, that only occurred in about 1-2%
13:02
of the people who were infected but so many people were being infected so many children were being infected and we were getting a high incidence of poliomyelitis. And so that disease can be stopped even though we don't have sterilizing immunity and may still replicate the virus. The same is true of measles, it will replicate in the oropharynx but it doesn't cause disease
13:21
unless it can get through the blood to the site where it's going to cause damage. Now the viruses where we have tremendous problems are the viruses that either change so that they can avoid the neutralizing antibody and the classical example is the human immunodeficiency virus that causes AIDS or the viruses that
13:41
change by some other mechanism such as reassortment or recombination as occurs with the influenza viruses. Influenza viruses grow in birds and humans and they have a packaged gene, a segmented genome which if you infect a cell simultaneously with two different influenza viruses will repackage and give you a new
14:00
virus. So if you happen to be in a situation where you've got a duck and a human being living closely together and the human being gets infected with the human influenza virus and also with the duck influenza virus you can get a new influenza virus out again which will be part human part duck and could cause an enormous epidemic in fact
14:21
and this is one of the things that we're absolutely terrified about in epidemiology because influenza will kill large numbers of people an influenza is a highly lethal disease. And that's also part of the problem is that influenza is a superficial mucosal infection and this is the other area where we have great difficulty
14:41
in providing sterilizing immunity. Though we can provide sterilizing immunity for something that goes through the blood it is very difficult to keep enough antibody at a mucosal surface to stop the virus from actually getting in. Antibody is secreted through here by various means but we need a lot of antibody around
15:01
to get enough through in the mucosal area. Excuse me, I didn't mean to blow into the microphone quite so bad. We'll see what happens with this soon in fact because a vaccine that is going to try to prevent mucosal infection is currently under
15:21
trial. That is the human papillomavirus vaccine. Papillomavirus is the cause of cervical cancer in women and the vaccines are currently under test to see whether we can actually stop that virus from transmitting. That would be an enormous advantage of course if we could. Now for the rest of this discussion I'll talk about cell-mediated
15:42
immunity. Cell-mediated immunity is to do with getting rid of the virus infected cell. Viruses are obligate intracellular parasites. They can only grow within living cells. One virus particle will get into a cell, you'll get millions of virus particles out. It's essential if you're going to terminate the infectious process to get rid of the virus infected cell,
16:01
to get rid of the factory that's producing the virus. This is actually the virus I showed you before, the parainfluenza virus, one virus, electron micrograph showing the viruses in the cell. We have to destroy that cell if we are to end the infection. And the way that we do that is by cells that are particularly programmed to destroy the virus infected
16:21
cell going from the vasculature into the tissues. These are highly migratory cells, the T lymphocytes, they migrate through between vascular walls and you can see some of the blood vessel lumen migrating through and then emigrating, in this case into the central nervous system. And what they do is induce
16:41
apoptosis in the virus infected target cell. They in fact trigger the cell suicide pathway and the cell in fact self-destructs. As we know altruistic cell death is a basic feature of cell biology and as we understood over the last few years in fact and this is what the T cell does. It causes the virus infected cell
17:01
to suicide. This works through the classical cell death pathway, the same one that's used by FASS, FASS ligand interaction. In the case of the T cell, the T cell carries large granules in its cytoplasm which include two sorts of molecules that are involved in the induction of this process. One is the perforins which
17:22
make a channel in the membrane of the target cell at the point of interaction between the T cell and the target and the other is the granzymes which are serine esterases, various types of esterases which are then thought to pass through those channels and induce and trigger the cell death pathway. This is thought to operate through the caspases and it induces
17:42
the latter part of the same pathway that is induced by FASS-FASS ligand interaction. This causes rapid cell death. The cells are dead within about five, certainly dead within five or six hours after the induction of this pathway. When T cells induce cell death via the FASS-FASS ligand pathway, it seems to take longer but it's also
18:02
effective and also works we believe in viral immunity. Now of course if you're going to have cells that are going around the body killing other cells you want to have that under very precise control because you don't want to have promiscuous killing, you don't want to have large scale tissue destruction. These are very powerful cells. They will kill one cell then they will go on and kill another
18:21
and kill another and kill another. So they have a very great potential. But they will only kill the cell which is specifically modified by the process of infection. They will not kill the cell next door which is not modified. And the way that works we all now understand is when the virus infects, of course its main motivation, all a virus cares about
18:41
is to produce new virus particles and to keep infecting other individuals. That's how it survives in nature, that's how it evolves. When the virus infects it uncoats, it exposes its nucleic acid and it makes new protein. That protein is always made in excess. Some of it is chopped up through the proteasomes, it goes into the Golgi and the endoplasmic reticulum
19:01
through the taps, into the endoplasmic reticulum through the taps where it associates with the nascent strong transplantation molecules, the class 1 MHC glycoproteins, major histocompatibility complex glycoproteins. These are called major histocompatibility complex glycoproteins because they were discovered by people in Joe Murray's lineage
19:21
who were working on transplantation. We knew that we had molecules that were involved in transplantation, that were specifically recognised in transplantation and for many years after we understood a great deal about transplantation we had absolutely no idea why those molecules existed. Why would God design a system whereby a kidney from one person was rejected
19:41
when it was given to another person? There was no evolutionary reason for that. We don't normally have kidneys transmitting between people in nature. Was it just there to frustrate the transplantation surgeons? Some of the physicians thought that was probably a reasonable motivation because they think transplantation surgeons are pretty appalling but we know that transplantation
20:01
surgeons are actually one of the few people that can really help us when we get in real trouble. And it turns out what these so-called major histocompatibility complex molecules are for is in fact to signal that our own self has been changed by carrying peptides from a foreign source to cell surface, in this case from the virus.
20:21
So we call it major histocompatibility complex and we call it transplantation but what it would have been if we discovered it another way would be called the self-surveillance complex or the self-monitoring system. It's just that it's historical that it was discovered in that way and it has that terminology and science is full of that sort of situation where
20:41
you have a terminology which constrains you to think in different ways that is actually determined historically and has nothing to do with the real biology of the system. For instance if you take the immune system there are all sorts of proteins which are enormously important that are called interleukins which says that they were discovered originally in leukocytes
21:01
and it says well these are something to do with the immune system. When we then turn around and we find those interleukins expressed in the brain the questions then asked is why does something that's in the brain that's in leukocytes get expressed in the brain? Why are we expressing an immune system molecule in the brain? Well it's probably the other way around. Evolutionary
21:21
the brain is a lot older than the immune system. There are organisms that have brains and don't have immune systems. The immune system has stolen something from the brain rather than the other way around but thinking the way that things are described conditions your perception of them. It's one of the things we have to be careful of in science as we try to think conceptually
21:41
about complex systems. We don't get locked into rigid intellectual frameworks which is a problem for all of us I think at times. So this is what the T-cell recognizes. It recognizes very specifically an 8-mera or 6-mera or 7-mera peptide from the virus presented in the transplantation molecule which now makes the cell look foreign. These peptides as I said
22:01
can come from any part of the any protein of the virus, internal or external. They can come from polymerases and ribonucleotide reductases it doesn't matter what it is as long as it's from the virus and it's not self. They have the potential to be recognized. We tend to have immunodominant peptides in a large virus. We may have say
22:20
one or two peptides which are normally recognized in association with a particular transplantation molecule. A lot of people are working that out. Robert Hubert told me he thinks from their studies that this is working at the level of the proteasome but there's still a lot of questions to be answered in terms of antigen presentation and processing. Now this is what the thing
22:41
actually looks like. This is the tip of the class 1 MHC molecule. This is the peptide. I think when we saw these pictures which was 1996 this is from Ian Wilson's laboratory at the Scripps. We were all surprised at just how little of the actual non-self is expressed on the surface of the this is the tip of the MHC molecule
23:01
is actually expressed. The T cell receptor sees both this and that. So the T cell receptor is seeing both self and non-self and it interacts also with the self major histocompatibility components. When we get into discussions of thymic differentiation and so forth
23:21
we get very much into considerations of this dichotomy. Now that's one sort of picture of the peptide in the MHC molecule. This is actually another picture. This is actually a monument which is at Memphis International Airport. Though I'm Australian I work at St. Jude Children's Research Hospital in Memphis, Tennessee.
23:42
Memphis, Tennessee does not regard itself as one of the great intellectual centres of the world. It's well known for Elvis and for pork barbecue. But it doesn't have great academic pretensions. So they were enormously pleased when a typical southern American boy like me was awarded the Nobel Prize. So they
24:00
decided on erecting a monument which they did in the departure area of the airport. This is a 12 foot high stainless steel cenotaph. Memphis was the ancient capital of Egypt. And here we see a hologram of the class 1 MHC molecule carrying peptide. They claim it is the first hologram monument in the world. And I don't
24:20
know what other actual statues of protein molecules there are in the world. But Max Perutz will know that and I will ask him afterwards. And he can tell me. Fortunately it doesn't have a picture of me on it. So I can walk through the airport and be absolutely anonymous. But it does have a plaque saying something about me and about zinc and algal and so forth. Now
24:40
they wanted to attribute that structure to zinc and algal and to me. It's not our structure. It came along after our discovery. It was actually the structure of Pam Walkman and Don Wiley and Jack Strominger at Harvard. And I said that if you attribute this structure to zinc and algal and to me, that will be plagiarism. Not only plagiarism. It will
25:00
actually be monumental plagiarism. And so I said that Don Wiley has some affiliation with Memphis. In actual fact his parents live in Memphis. So he's now described on this as a native Memphian Don Wiley. In actual fact he has never lived in Memphis in his life. But that makes the very little difference. Once one
25:20
gets outside the sort of scientific laboratory world that we normally live in and you get into the world of the media, you find you're in a totally different situation where truth is extremely relative and doesn't matter too much. With due apologies to the gentlemen of the press I'm sure who are all from the scientific press that are extremely
25:40
careful. I've had a lot of public debate and so in Australia after the Nobel prize and I discovered that one can have some extraordinary experiences with particularly the print media. Now respiratory infections. For the rest of this I shall talk about some experiments
26:01
and just about what happened in a fairly simple respiratory infection. As you can see respiratory infections are a major problem. They continue to be a major problem. This is second world war British propaganda where they're trying to stop people from infecting each other and thus decrease production. The influenza epidemic, the
26:21
world's worst virus infection epidemic before the AIDS epidemic was the 1918-1919 influenza epidemic. It came at the end of the first European war. It contributed considerably I think to ending that war because people were dying in the trenches on both sides. It was called the Spanish influenza. That was
26:41
because the Spaniards were the only people who would admit to having it. They weren't involved in the war so the Allies weren't going to admit to having it and the Germans weren't going to admit to having it so it was blamed on the Spaniards. Now it actually killed worldwide somewhere between 20 million and 40 million people. That's many many more people than the war
27:01
killed and it killed them all over the world. When I say between 20 million and 40 million it certainly killed 20 million and it's thought it may have killed another 20 million in India but the British were controlling India and they didn't bother to count them. So it was an enormous epidemic and there's no gap even though we understand how influenza works now
27:20
they didn't know what influenza virus was at that stage and we know we can make a vaccine against it. It still will take us probably six months to get large quantities of vaccine out into the population certainly three months, three to six months. This killed people all over the world before there was jet transport and
27:41
while there was still quite strong quarantine measures and acceptance of quarantine measures which no longer really work and so we're all actually rather frightened of another influenza epidemic which is why there's a very strong virus watch program going on in China at the moment. One of my colleagues Rob Webster is setting up activities in Hong Kong because a lot of these
28:01
viruses tend to come out of Asia and we're watching all the time to see for emerging influenza viruses because we think that apart from AIDS this could be something that would give enormous problems. There is no way you can protect yourself against a respiratory infection despite this cartoon.
28:21
You can't stop yourself getting it. There's no behavioural change that will stop you getting it. The only thing that worked in this epidemic that stopped a population getting the infection was in western Samoa which was controlled by the United States Navy and they shut up western Samoa. They didn't let anyone in.
28:40
They didn't let anyone out. It was just like Alcatraz Prison but nobody died. In eastern Samoa which was controlled by Democratic New Zealand lots of people died. This is the influenza virus electron micrograph and what we're going to talk about with this is mouse experiments. Now the basis of all immune responses of course is clonal expansion and
29:02
differentiation. There are relatively few lymphocytes which bear the receptors that recognize say the influenza peptide or the influenza protein if they're B cells. What the immune response is is a process of proliferation. You select from those very few lymphocytes they proliferate and then we end up with large numbers of lymphocytes. They proliferate and differentiate.
29:22
Some differentiate to be effector cells some go on to become memory cells. We talk about a primary response this is one that the individual has never encountered before, the first time we encounter influenza or a secondary response. We've encountered this infection before we've got better, we're okay and then suddenly we get something like that again.
29:41
What will happen then is we will have expanded numbers of lymphocytes which can react to that infection we will get a more rapid response. These are memory T cells and we're talking about a T cell response. The characteristic of memory T cells is there are many more of them than there are primary T cells and they're also partially differentiated. Probably their chromatin has changed and so forth
30:02
and they turn on much more quickly and they will respond much more quickly. So we will get a more rapid response. But as I will show you there are limitations to this. These responses occur in the lymph nodes which is the part of the body that's specialized for immune responses to develop. It's a sort of a nurturing environment that allows all the various components
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of the immune system to come together to get this response going and the first thing will happen in any virus infection if you get a respiratory infection is the lymph nodes will swell and you will have swollen lymph nodes in your neck if you've got a respiratory infection. What is happening there is this nonspecific recruitment of white blood cells that are in the blood and the lymph
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into those particular lymph nodes that are infected and it's thought that some of the cytokines and so forth that are secreted by virus infected cells particularly alpha interferon draining into those lymph nodes and causing this selective recruitment to the site where the immune response needs to occur. And so what happens is something like this.
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This is a cartoon of a respiratory infection in a mouse. We infect the mouse intranasally and the mouse gives it the virus down the nose, influenza virus. These viruses grow only in the superficial epithelial cells of the respiratory tract. They only cause productive infection in that site is that they only make new virus progeny in that site. The reason for that is
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that they're the only cells that have an enzyme which cleaves one or other of the surface proteins of the virus. With influenza it's the hemagglutinin protein. So you can get a defective infection in other cell types but you don't get virus production. You need that enzyme to actually give you a productive infection. So even though these cells only infect that
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superficial layer of cells they're still highly lethal. They don't generalize, they don't get systemic, they don't infect any other site in the body but they're still highly lethal simply by destroying the respiratory tract so that we no longer breathe. Some of the virus will also go into dendritic cells which are specialized antigen presenting cells that
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are particularly apparent in the upper part of the respiratory tract. They're all around the body in various forms but there are large numbers in the respiratory tract. They will carry virus or protest virus down to the lymph node where the immune response will then develop. That will take six or seven days and then after that we will start to see the T lymphocytes coming out and the B lymphocytes
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as well and going into the, particularly the T cells, going into the virus infected respiratory tract. They'll come out into the lymph, into the vena cava and then into the blood stream and they will escape more or less on a stochastic basis into the virus infected respiratory tract. Here they will become fully
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functional killer T lymphocytes which will bump off the virus infected cells here and gradually the infectious process will resolve. If we look at these inflammatory sites which we obtained by Broncoalveal Olavage, you will see the term BAL in some of the slides, we'll find about half the cells that are there are monocyte macrophages
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and about the rest, about 60% of the rest are CD8 T cells, the killer T cells and about 30% are CD4 T cells, the helper T cells. After about 10 days the virus will be eliminated, the whole thing gradually resolves and the individual gets better. Though people can actually remain quite unwell for some time after
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respiratory infections and it may be due to continuing cytokine release. Now, these are the inflammatory cells we obtained by Broncoalveal Olavage and as I said the CD8 T cells actually work in this infection by direct killing. They can work through killing by that perforin mechanism or killing by the FAST mechanism. With
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influenza CD4 T cells, I haven't said much about them, but CD4 T cells can also control the infection but they do that by providing help for the antibody response. The essential point about the CD8 T cell is it must make contact with the virus infected target cell. I've done a lot of experiments to show that over the years. I've been very emphatic about it and someone gave me this slide.
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This is not to discourage discussion with the students later. Probably bigger and stronger than I am anyway. Now, memory is established and it's enormously important. It's been known for many, many years and it was first described by Thucydides, I think he spelt wrongly, writing about the Peloponnesian
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Wars. Only those who recovered from the plague could nurse the sick because they couldn't catch the disease a second time. The altered status is specific. This is the basic dogma of immunology, so he was somewhat ahead of any immunologist that I know of. That's Salvador Dali's idea of memory. Of course he's thinking about neurological memory.
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You can see the floppy clocks and so forth. He was influenced, I think, by the sort of intellectual discussions that intelligent people used to have at that time. They used to talk about Einstein's relativity theory and so forth. That's the floppy clock. Now they will probably discuss Dallas or the tennis. Here you can see an immunologist, this sort of floppy, spineless
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looking creature. The way we thought about memory until very recently is that we had from techniques which are really rather imperfect, we did not have methods for directly measuring the number of antigen-specific T cells. This was a considerable problem to us. All we could do was take lymphocyte populations, put them in little
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culture wells and expand micro clones of T cells and try and count those clones. We had to proliferate those T cells over six or seven days to get enough T cells to read out in our assays. This required that lymphocytes go through at least 10 or 12 cycles of division in the tissue culture wells and we never quite knew whether we were measuring all the T cells.
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Still from that protocol, we developed this sort of idea of memory which is basically correct, though some of the numbers may actually be wrong. That is initially in the immune response, you get enormous expansion, a great over-production of T cells and then some of them stick around more or less forever as memory T cells. They may be less activated
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as time goes by, they may be less readily re-stimulated but they are increased in numbers for a long time, certainly for the life of a laboratory mouse. We know much less about long-term memory in humans which is a problem for us because of course humans live about 35 times as long as a laboratory mouse and I'm not at all sure that one year in the life of a laboratory mouse
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is equivalent to 35 years in the life of a human. Now this is the influenza virus and as I said the T cells, they vary tremendously due to this reassortment or recombination process but also because of immunoselection in the presence of antibody. But the T cell response can be directed against these internal proteins
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and what I'll talk about is a response that's directed against an internal nuclear protein peptide which is actually very conserved between the different influenza viruses. That means we can infect mice sequentially with different influenza viruses which will not be recognized by neutralizing antibody, will not be cross-neutralized but they will share the same T cell
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response element, so they will then share the nuclear protein peptide. Now we can study that response now extremely quantitatively. This is studying it by cytotoxic T cell activity, the killing activity. Here you see the primary response, slower and of smaller magnitude than the secondary response. This is an old slide, it goes back to the late 1970s
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but we can now study that in a very very quantitative way indeed because we can now actually stain the T cell, the T cell receptor that recognizes the peptide MHC interaction. And the way we do that is by the use of tetramers that were developed by John Altman working in Mark Davis' laboratory in Stanford. The first papers published using these things were published
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in 1996 and what he did was take MHC molecules plus peptide and then link them with an avidin core and this gave us something with sufficient avidity or affinity to bind to the antigen specific T cell. So now we can directly visualize the T cell, something we
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could not do before that or directly visualize the antigen specific T cell using the flow cytometer. And these were labeled with various fluorochromes and then as we pass them through the beam of the flow cytometer we can actually stain the immune T cells directly and actually measure their numbers. And I'll use that just to show you the difference between a primary and a secondary T cell response.
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This is a primary response to a Hong Kong influenza virus H3N2, hemagglutinin 3 neuraminidase 2. Here we're seeing on this axis we see staining for the tetrameric reagent that recognizes the nucleoprotein peptide which is immunodominant in this response in an H2B mouse.
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And here's CD8. And here's days after infection. These are bronchoalveolar lavage cells which are washed out of the lung. Three days after infection we see nothing. Five days we see nothing. Seven days we see a few cells. The virus is eliminated between day 7 and day 10. And here we see about 12% of the CD8 T cells
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that we isolate from the bronchoalveolar lavage. That's this top right hand corner staining both with CD8 and with the tetramer are specific for the virus. Here you see the difference with the secondary response. These mice were primed eight months previously with this other influenza virus, the H1N1 virus. So they
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share the peptide that's recognized by the CD8 T cell but they don't share the surface components recognized by neutralizing antibody. And here you see the difference between a primary and a secondary response. Here, nothing on day 3 which is a bit disappointing for people who are trying to make CD8 T cell vaccines but here we see the T cells on day 5.
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Day 7. And by this time, 70% of the CD8 T cells and the bronchoalveolar lavage have that specificity. This was quite extraordinary to us. We had absolutely no idea that this was actually the case. We've been able to confirm that by different techniques. I won't go into that. Can we skip the next two slides, please? Not that. Go on.
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That's just another technique showing the same thing. Go on. And here we see what happens. This is the virus growing in the respiratory tract of a primary infection. You see the virus we isolate from the lung and then it's eliminated. These are the virus specific CD8 T cells coming up here. They're responsible for the elimination of that virus. This is the
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secondary response. Early on we get just as much virus growth in the respiratory tract then the virus is eliminated more quickly and here we see the much bigger secondary response. We get enormous numbers of lymphocytes in the lymphoid tissue, in the spleen and so forth in the secondary response. This is the primary response. We can just detect them with our flow cytometer in the primary
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response. Here's the spleen. This is the secondary response. Massive numbers of T cells. This is creating enormous numbers of questions for us as we try to understand the immune system. Here we have in a secondary response which a virus that grows only in the respiratory tract causing 25% of the CD8 T cells in the spleen to be of a single
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specificity. As it was we had enough difficulty understanding how it was that we covered all the bases with immune responses. How do we have enough lymphocytes to really recognize all these various infectious agents and when we had an immune response how do we fit them all in? The immune system doesn't basically
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change in size. It's subject to homeostatic control just like any other organ in the body. But here now we have 25% of the CD8 T cells in the immune response with a single specificity. This is now rather typical of virus infections. As 25% immediately after the infection, by 100 days after the infection we're still around about 8%
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of the T cells with that single specificity. It makes it even more difficult for us to understand what is really the most difficult problem in immunology. That is understanding homeostatic control. It is attracting a lot of conceptual activity but it's difficult to actually work on
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because we're dealing with populations of cells which are dispersed around the body they're in the lymphoid tissue, they're in the blood, they're in all sorts of different tissue sites and it's really quite a challenge to understand how this whole thing works. Especially when we're getting these large numbers of T cells specific for a single entity. And so that simply summarizes
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what I've said. I'll leave the story there. This is Dali's last word on the program. I'm not sure what he's symbolizing there. I think all these sort of arrows and things here are actually Jack and stat molecules and they're showing that the signal transduction people are taking over the field anyway as they are with everything else. And this is
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the last word on Burnett actually. Burnett is because it's his centenary, he's actually traveling around the city of Melbourne where he worked in the Walter Eliza Howell Institute on the side of a tram car. And he shares that tram car with Howard Flory, another scientist who was an Australian scientist who awarded the Nobel Prize
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who was 100 years old last year if he'd lived that long. Here we have Burnett I don't know how well Flory and Burnett got on but they're on the same tram car. Here we have Burnett saying science to me is the finest sport in the world. Not surprisingly this doesn't really resonate with the Australian popular culture
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which is totally sport obsessed. But still, and also most of the scientists in Melbourne don't seem to realize that Burnett is rotating around Melbourne on this tourist tram. I talked there last year and pointed out that this was the case and none of them had actually seen the tram car though it's been going around Melbourne for a year. Which is
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just a point if you're thinking about orienting your career so that you win the Nobel prize don't actually believe that you're going to achieve any permanent glory whatsoever. The best that you're likely to achieve in the popular sense is to end up on the side of a tram car and that only very briefly. They've recently taken Faraday
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off the British 20 pound note I think it was and replaced him with the musician. The reason that was given was that Elgar had a much more spectacular moustache which would be very difficult to counterfeit. Thank you.