 It is my very distinct honor and special pleasure to present to you at this time our next conference speaker, Dr. Robert Gallo. Dr. Gallo is a most esteemed investigator of biological mysteries. His interest in the area goes back to childhood and a relationship he had with a pathologist who used to fascinate him with medical detective stories. So as a child then he knew this is his kind of work. He wanted to do that. So when the appropriate time came he went off to college, got his BA at Providence College, went on to Jefferson Medical College and later internship and residency work at the University of Chicago. While he was still in school his gifts as a medical investigator caught the attention of National Cancer Institute and they hired him right out of residency as a clinical associate and three years later promoted him to the post of senior investigator in the laboratory of human cell biology. The rest has been a stunning record of achievement and service to the betterment of humanity. In the area of cell biology, the study of white blood cells, possible viral causes of cancer and leukemia and immunological investigations of the HTLV virus which eventually led to the intensive study of AIDS and the discovery of the AIDS virus in development of the AIDS antibody test. The circumstance I'm sure many people know also plunged Dr. Gallo into also the human realm of international controversy and intrigue. Along his path Dr. Gallo has accumulated an array of prestigious awards and prizes for advancement in scientific research including the Albert Lasker Award which I believe Dr. Gallo is the only person to have won that award twice in 1982 and 1986. And there are other prizes and awards too numerous to mention. You can see some of them listed in your program and there are still others. He is by the calculations of the Institute for Scientific Information the most widely referenced scientist of the 1980s with over 23,000 citations in medical literature. He's still with his original employer back there at the National Cancer Institute at the National Institutes of Health in Washington where since 1972 he has served as the chief of the laboratory of tumor cell biology. He's going to talk to us today about human retroviruses. Won't you welcome please Dr. Robert Gallo. I'd like to begin by thanking the organizers for inviting me. I'm honored to be here with you. I want to particularly thank Chaplain Ovi and Dr. Lemert who I think are the major organizers and I assuming did most of the work with their staffs. And naturally I want to thank Rich for introducing me and for being not my shepherd but maybe my guardian angel also keeping me from getting lost all the time. I'd like to talk to you about the AIDS epidemic from the perspective of a laboratory scientist who began as a MD sort of. And I'd like to do it by having some reflections to the past and also to the future but with concentrations for the work we're doing now namely of course present. At the beginning of the epidemic all of you may recall was 1981 when the disease was first recognized on both east coast and west coast of the United States among gay men. That epidemic however had started substantially earlier and wasn't recognized because it took so many years for the disease to become manifest in the people who were infected. When this happened many people most of us in the scientific community were a little shocked, surprised. It was unexpected. We felt that microbes that cause major epidemic fatal disease were really things of the past maybe things for non industrialized nations. We had after all learned a lot about sanitation, we had antibiotics for bacteria, chemicals for some other kinds of bugs. We learned how to avoid such things but rereading or reading for the first time the nature of epidemics of the past. We can see that even in antiquity without the so-called sophistications that we have now epidemics could disappear for long periods of time. There is suggestions that in the Greek and Roman period there might have been 100 and 200 years without serious epidemics. So we're reminded that microbial epidemic serious disease can come out of the blue it seems, come and go sometimes, sometimes come and apparently not go. So let's go back to some aspects of that early period and say first of all that it wasn't new to have a new epidemic. It also wasn't new in the way society responded. There were a lot of criticisms about the ways governments and society first accepted the AIDS epidemic, but looking at previous epidemics it seems to parallel exactly the same kind of emotions, disbelief, then belief fear over reaction, hostility, blame and failure to understand the science by majority. There was however a few things that were new. One thing that was new were changes in the society, a point I'd like to come back to in just a moment, changes in society which made a rare infection of human beings become global and relatively common. There's another change, or excuse me, something else that was new and that's the nature of the microbe that causes AIDS. We can face something exactly like this before and I believe it is not just the AIDS virus, but collectively we can look at a group of viruses, not even viruses all of the same general kind that have become increasingly important in this century as time has gone by. I've tried to summarize their properties in the first slide that I'll present to you. Most of us growing up would think of a virus as something we catch easily, get rid of fairly easily and don't usually get seriously harmed from it. But the viruses that we're more and more concerned about are viruses that we can summarize this way. They're new, or if they're not new, at least they are newly discovered. The second point is that they're difficult to find. Most viruses that we are accustomed to think about from the past are replicating in a lot of cells and are not so easy, excuse me, not so difficult to isolate or not so difficult to detect. But the viruses of current importance more and more are those that replicate inefficiently or have very few target cells and are often invisible to the, not just the naked eye or a microscope, but even to the electron microscope. They're not just small, they're absolutely invisible at certain times in their replication cycle. They're difficult to transmit often, difficult to transmit because they replicate not so efficiently. There's not so many of them and all of biology is quantitative and to infect a cell takes a certain number of virus particles that maintain the ability to infect. Many particles within a group of viruses will be defective. Some will be defective because they've been physically partially destroyed. Others will have their genetic information partly defective. So there has to be a reasonable number of virus particles to have an infection. And these viruses, as I said already, also have special target cells. Thank goodness that the AIDS virus cannot infect the common cells of the nasal or oral pharynx or we would have a casually transmissible agent that would certainly be the end of humanity. The fourth point is that they have a very long latency period. This means from the time of infection to visible disease as many years often. Sometimes as long as 7, 8, 9, 10 years and sometimes even still longer. A group of them that I'll talk about just as a digression almost usually takes 10 to 30 years to produce its disease. Imagine if you don't have the virus isolated and you don't have any way to check for it because you don't have specific probes. How do you follow an epidemic? You look for disease. How do you follow disease when it's poorly transmitted among a group and takes 10 years to become manifest? It becomes a nightmare. The fifth point is that these are infections from the time of birth until death. They're lifelong infections in many of the cases that I'm referring to. That's certainly true of course of the AIDS virus and as far as I know for any virus of the same general category which we call retroviruses. They can be transmitted to others during that long period from the time of infection to the time of birth. Point number five. This is especially important point. There are textbooks of clinical microbiology that state even today because they were written before we understood many aspects about AIDS that it is impossible for a microbe to cause a global epidemic, a pandemic if the microbe isn't easily transmissible. But we know with the AIDS virus that's no longer true. Everywhere in the world there is infection with HIV now except in Antarctica. So it became relatively rapidly global even though it's hard to transmit and requires intimate contact with body fluids and usually a reasonable amount of body fluids. What explains that are two things in my mind. The first is that the virus is present so long as an infectious agent. With other viruses, most viruses that we think about, the time you can infect another person may be a day, a week, a month, but not 10 or 20 years. The second reason again is the changes in society which I said I'd come to in just a moment. The sixth point is that many of these microbes, these viruses are causing serious disease. Diseases of the brain and spinal cord, malignancies, cancers, related abnormal cell growth as well and of course serious immune deficiency. They become increasingly important and examples of them include all the human retroviruses but especially the AIDS virus, papillomaviruses, some of the hepatitis viruses, probably viruses we haven't yet discovered and in some respects even herpes viruses, although the herpes viruses are certainly more readily transmitted, they too set up persistent and long time infections. I won't talk about it today but in fact, six years ago my colleagues and I found a new herpes virus called Human Herpes Virus 6, HHV6, which is becoming more and more important as time goes by, although at the beginning we thought it was just going to be a passing fancy for us. Now how did the AIDS epidemic begin and where did it begin? No one can say with absolute certainty but logic and a lot of circumstantial and indirect evidence strongly indicates that this virus originated like much of life from Africa, probably from equatorial Africa. One of several reasons is the presence widespread particularly in the sarcopethicin family of monkeys, of a related virus known today as Simeon Immunodeficiency Virus or SIV. Virus present in these monkeys, SIV, is very closely related to the human immunodeficiency virus, the HIV called HIV2. For reasons no one understands, HIV2 is relatively limited to West Africa and despite extensive study of its molecular biology, HIV2, the reason HIV2 is spreading less rapidly and causing AIDS with less efficiency remains unknown. But I repeat, virus is present in many of those monkeys is as much an HIV2 as different isolates of HIV2 are from different people. HIV2 is a Simeon Immunodeficiency Virus. Now what about the real AIDS virus, HIV1? The SIVs present in these monkeys are significantly different, 50, 60% of their genetic information is quite different from HIV1 but in the last few years there's been at least a few isolates from chimpanzees of a virus that is significantly closely related to HIV1 and is probably the ancestral origin of the virus in human beings. I believe these viruses have been present in human beings for a long time. Not as long as the leukemia viruses that I'll talk about in just a second as one modest digression. They've been present in man for very, very long time. We can't say when these viruses first entered man, one can guess, maybe several hundred years, maybe a thousand years, it's impossible to condone. But the epidemic is very known. The epidemic begins sometime probably in the 1960s, early 1960s, and it's really epidemic in the 1970s and certainly we're seeing disease, of course, by the 1980s. What caused a rare virus to spread like this? Earlier I said I wouldn't come to the point of changes in society. The evidence would suggest that this was probably an infection in rural or bush populations and that people died with their disease. It didn't spread, it didn't become epidemic. Post-World War II, the development of commercial travel, tourism, the airplane. Big contacts of people who didn't have so many contacts before. It's possibly one explanation, increased sexual promiscuity, intravenous drug addiction, blood supply and blood products moving from one nation to another nation. Specifically in Africa perhaps the departure of colonial powers in the upsetting of old ways and inability to go back to old ways combined with famine and war and population movements to large cities and increase in prostitution. It is reasonable that this began, the epidemic that we call AIDS today. Now I'd like to move to where we are today in the epidemic. The slides that I'll show you, there will be three, are a little bit outdated. They're one or two years old now. These look at numbers of AIDS. Not of infected people, but of, what is it? No, this is, I'm sorry. This is the distribution of HIV infection in the world. The number of people with infection right now in the world is something like around 10 million. It's probably more. It's under reported. It can't be reported in certain countries of the world accurately. It is estimated that this number will increase by the year 2000 to somewhere between 50 and 150 million people. So that's the pattern today. This is the projected estimates from the Center for Disease Control in Atlanta, Georgia. And it looks at different areas of the world. Let's look, for example, of our own area, industrialized countries. And you can see that there's a relative plateauing in the rates of infection predicted for the coming years. That is predominantly due to the decrease rate of infection among gay men in the industrialized nations. But if you took subpopulations out of that, you'd see a striking increase in the rates of infection. For example, in so-called Hispanic populations in big cities of the east coast of the United States, there is dramatic rise in the children of such people, the babies born to infected women. So in some populations in the United States, there's dramatic rise. But overall, the rate seems to be about plateauing. All of you know that increasingly it becomes more and more talked about as spreading also by heterosexual intercourse. But that is not a surprise. You all know that that was known from the beginning in other places in the world and especially in equatorial Africa. Look at the dramatic rise shown in yellow in sub-Saharan African countries with apparently no end in sight. And look at the sudden jump in the white over here from nothing. In 1986-87, going up like this in some of the southern eastern Asian countries, especially Thailand, also the Philippines, and most recently India. And you can also see a steady rise in the so-called Latin American countries. Another interesting way to look at the epidemic is the way the virus is now known to be spread, or estimated to be spread, again from center of disease control in Atlanta estimates. And slides also list the relative efficiency of infection by the virus. So we can see that the bulk of infection today is occurring by sexual intercourse, 80%. And the majority of that is vaginal sexual intercourse, 70% of the total. Another significant increase in infection is of course by intravenous drug abuse, which is estimated to be 5-10%. Healthcare workers at risk, a low but significant number. Blood transfusions are disappearing with the development of the blood test. And increasing infection perinatally from mother to child, the majority of that occurs during the process of delivery. It's interesting and I don't understand it, maybe some obstetrician will explain it, but in twins when they're born, the first twin has about a four-fold greater chance of being infected than the second twin. The efficiency of infection is of course, if you get blood transfused with the virus, the efficiency is incredibly high. It's probably close to 100% because of ghosts and it gets into your bloodstream. Perinatal infection is also highly efficient. Sexual intercourse is not so efficient as you can see, 0.1 to 1%. That means in any given time it's about a 1 in 100 to 1 in 1,000 chance with an infected partner. Of course it depends on the partner. Long ago studies, for example, by Frank Plummer in Canada demonstrated that you're more infectious if you have venereal disease of another kind. For example, a man who has gonorrhea has more white blood cells, pus cells in his semen, which can be loaded with HIV. He would be more able to transmit virus sexually than a person who doesn't have gonorrhea. In a woman with venereal ulcerative disease has much greater chance of transmitting virus to a male sexual partner. I'd like to now move away from this discussion of the epidemic as such. Well, actually before I go further, I want to say one thing about the blood test. You hear a lot about the blood test, Rich mentioned it in his introduction. I want to mention to you what the blood test does and what it exactly is. We can't isolate the virus from every person that's infected. That would be logistically, economically, technically not feasible. Antibodies present in the serum of people can be detected with great sensitivity and accuracy. And with a retrovirus, defining an antibody doesn't mean you're protected. Rather, we knew from the start from experience in animal retrovirology that the presence of antibodies in serum is an indication of active replication of the virus, the presence and maintenance of the infectious virus. So that's how we test for the virus by testing for antibodies as telltale signs of infection. The development of the blood test did three things at once. It allowed the blood supply in the world, I shouldn't say the world, but in a lot of places it's still not done, but in the industrialized world, to become safe. So it saved lives. Secondly, it allowed the epidemic to be followed immediately, carefully for the first time, changing the nature of the search. And thirdly, it was the most important parameter in demonstrating a positive link of HIV data. During that period, we tried to correlate the finding of antibodies with the ability to detect and or isolate virus. We carried that out for a while, finished it, and now one can stay in terms of monitoring the epidemic with a highly accurate, predictable blood test for infection and for the development of AIDS. Dr. Bernazerath, in his superb talk this morning, mentioned that in some respects we were lucky when the epidemic appeared. I agree with that, obviously for a lot of reasons we're unlucky, but from the scientific point of view we were extremely lucky. Because of the developments in immunology between the 50s and mid 70s, we could understand what lymphocytes were to a significant degree. And as he said from work in his own institute, we knew the difference between some types of T cells and other types. Without knowing that, clinicians wouldn't have been able to tell us that this is probably a primary disease of a T cell that is called the CD4 positive helper lymphocyte. And also without developments in the mid 1970s where we learned how to grow T cells, we couldn't have isolated the virus. These and many other contributions from cell biology and immunology were principally responsible for rapid progress in those early years. There's another scientific series of advances that was also important. And those were the advances made in the study of animal retroviruses that began around 1910 and culminated sometime in the 1970s. And when we were about to quit looking for them in humans, we found the first one in humans that we called human T cell leukemia virus 1 and two years later a second one. And the experience with those human leukemia viruses, which are also retroviruses, was extremely useful to us in this early period of time. I told you earlier that I was going to make a small digression about AIDS and talk to you as a background about these leukemia viruses. This slide lists all the known human retroviruses that cause disease today. They are the HDLVs that belong to a subclass of retroviruses that are called ANCO for cancer. They're also known as Type C. The retrovirus of AIDS is called the lenti retrovirus. It's kind of a misnomer. Lenti means slow, in Latin, I guess. And these are not as slow as the HDLVs, which take longer, usually, to produce disease. So a few things about HDLVs that will be useful when I come back to the AIDS virus. This is a picture of the human retroviruses. And here are the leukemia viruses at the top, HDLV1 and HDLV2. And here are the AIDS viruses at the bottom. When you're lucky, when you have a lot of virus, when you have a cell line with virus production, you can distinguish them morphologically because the center, what we call the core, is more compact and cylindrical in the case of the AIDS retroviruses in comparison to the leukemia viruses. Now retroviruses have a name retro for a reason, of course. They're viruses also from the Latin, which simply means poison. Things you couldn't see that produced disease. We can now see them by electron microscopy. The virus, of course, is smaller than other microbes. But the key part of a virus is that it is an obligate cellular parasite. It can't live or metabolize. It doesn't metabolize. It's not like a bacterium or a fungus. It has to live only within the cell. It may survive somewhere like on a tabletop for a while, but it can't do anything. It usurps the host's own cellular machinery to reproduce itself, and this is why targeting of a virus requires far more sophistication than targeting a bacterium with antibiotics. When you hit it, you're often hitting exact components of the cell. So there has to be more subtlety in antiviral agents or in modulation of the immune system to fight a virus after infection has occurred. A retrovirus is special and has the name retro because it reverses the usual flow of genetic information, as I'm sure most are all of you know. Its genetic information is RNA, but that's not unique. Polio, influenza, and other viruses are RNA viruses. But what it infects the cell, the RNA is, let us say, for the purposes of this talk converted into a DNA form. And that DNA form integrates into the target cell chromosomal DNA, which is relatively unique for this category of viruses. You can picture this by thinking of a thread cutting out the center and inserting a different color into the thread. They insert their genetic information after the DNA is open. And in that form, the virus can be silent in a targeted infected cell. You may never see it. You would see no evidence of viral proteins. You would see no virus particles. It could be silent. But the infection of that cell is forever. And when the cell divides, the daughter cells will carry the viral genes because they duplicate with the rest of the chromosomal DNA and are transmitted to the progeny cells. Infection of the cell, infection of the individual, then becomes lifelong. That is the key definition of a retrovirus, an RNA virus that makes DNA when it infects the cell that the DNA becomes stably integrated into the chromosomes of the target cell. Let us now, as I've been hinting, to go to the leukemia virus's HDLv1 and HDLv2. This is why it is called a dendogram. It just looks at the relative relationships of a large number of isolates of leukemia virus's HDLv1, excuse me, one in different regions of the world. When I show you this to point only to the middle, the chimpanzee leukemia virus is as close to some of the HDLv's of man as they are to each other, mimicking the story I told you about the monkey virus relation to the AIDS virus. In other words, monkeys are widely infected with a leukemia virus that we call simian, STLv1. But these are ancient infections of human beings. Isolated Melanesian tribes with no history of contact to the west, as for as long as we know to anybody else, according to my colleague at NIH Carlton-Guydeschek, have a significant degree of infection with HDLv1. We have no idea what disease it causes in such people that may be harboring these viruses for long, long periods of time. At the bottom, far removed from HDLv1, most HDLv1's is HDLv2. Currently we believe HDLv2 is predominantly an infection of American Indians. HDLv2 disease relationship is more problematic than HDLv1 and we won't talk about it further. When we depict the genes of a virus, we do it this way, it's relatively simple, retroviruses don't have so many genes. So this is the entire genetic information that if it becomes active, it's going to form the human leukemia virus, HDLv1. Animal retroviruses were known only to have three genes, gag, pole, M. Those genes make proteins that form the structure of the virus and a few special enzymes that we won't talk about today. All retroviruses also have these regions on the end called LTR. The light doesn't work well, but I think you can see LTR in each end. Those are not genes, they're regulatory elements that govern the expression of the genes of the virus. They also form the sites of integration into the host cell DNA. In other words, to the left of that LTR on your left would be cellular DNA, linked, covalently bonded to the LTR. With the discovery of human retroviruses, we learned about new genes that are shown on your right. With HDLv1 and 2, we call them REX and TAX. We now know it's even more complicated, but this is not yet published and I won't get into it. These extra genes on your right, REX and TAX, we now know are critical for the replication of these viruses. When they were discovered, this was unique. But as you will soon hear from me, we have almost exactly the same parallels with HIV. When the HIV is still more complicated and contains still more genes whose functions we don't know. One thing I've learned from virology is that a virus doesn't waste one nucleotide, let alone one gene. So every one of those genes are going to be doing something to favor the virus's survival, its evolution, its ability to replicate its ability to fool the immune system or whatever. And we're only scratching at the surface of these other genes in HIV. Let us now turn to what we would call the life cycle, or better, the replication cycle of these leukemia viruses. And what I say for them will be more or less true for HIV. This slide shows a cell on the left and a cell on the right, shown at the bottom of the circular as the nucleus. On the top of the left you see the leukemia virus coming to the cell where the outer knob-like structures we call the envelope will bind to a surface molecule of the target cell. The surface molecule for these cells, for HDLv1 that binds it is unknown. For the AIDS virus, it is a molecule we call CD4, and that's the first step of infection by the AIDS retrovirus, an interaction between the envelope of the virus and the CD4 molecule of the target cell. For the leukemia viruses, as I said, we don't know yet what that molecule is. Following that step, there's a fusion of some lipid material of the virus and the cell lipid bilayer. And then the guts of the virus, the core, is emptied into the cytoplasm of the cell, and the RNA shown in yellow is converted to DNA shown in red, and the DNA gets to the nucleus where it integrates. That's the first half. Now that cell may sit there silently and you may never see the virus again. But if those genes become active, move your eyes to the right, and the viral DNA is now being what we call transcribed by cellular machinery listed on the slide, something called RNA polymerase. And you remake viral RNA shown in yellow, which will come out and act as a messenger RNA to make viral proteins. Some of the RNA won't be modified or processed. We call it genomic RNA shown to your far right in yellow in the cytoplasm. That genomic RNA gets packaged at the cell membrane with viral proteins. And eventually the virus will form by budding and pinching off the cell membrane to complete the cycle. Now what I haven't mentioned is that those extra genes called TAX and REXREX that I showed you on the genome slide are involved in the nucleus and they're involved in subtle ways of regulating the expression of the viral RNA. The TAX gene makes a protein that allows the rest of the viral genes to be made. The REX gene makes a protein that allows the proper transport of RNA molecules across the nucleus into the cytoplasm. However, in recent years we have also learned that the TAX protein of this virus also activates cellular genes. Among them are cellular genes that lead to abnormal T-cell growth. So this viral protein that is necessary for the proper activation of the DNA form of the integrated form of the virus to remake viral RNA, also turns on normal cellular genes, a series of them. And so doing so, in doing so it leads to abnormal T-cell growth in most infected people at some stage of their infection. And over the course of years some unlucky people probably due to hyper T-cell proliferation will develop subsequent genetic changes that lead to T-cell leukemia. This same virus can cause a disease that mimics multiple sclerosis. In addition to leukemia it causes a neurologic disease particularly of the spinal cord. And here the mechanism is not known but the hints are that it is probably some form of an autoimmune phenomena or a number of observations I could not go into that argue in that direction. Neurologic disease from autoimmunity leukemia can direct infection and promoting abnormal cell growth. In the case of leukemia the viral genes will be found in every cell of the tumor. Let me summarize and get away from HDLB-1 with this slide. Number one it's an ancient primate infection. It's endemic in a few percent of populations in African people and their descendants. It's found fairly widespread in southern Japan and especially it's also in some South Pacific people and many of Americans we see more of what we call HDLB-2. Thirdly it causes disease in a lower incidence not like AIDS. Perhaps 5 perhaps 10 percent of infected people for lifetime will get a significant disease. The diseases however can be serious like leukemia and the fifth point they may provide useful models for understanding leukemia in general and some central nervous system disease. A blood test is now required in the United States and in Japan for these viruses and lastly perhaps not least we learned some technology from them that helped us with HIV. Now this is a cartoon of the AIDS virus. Now at the top again I show you the genome of the virus this great line of different segments. Here again you see the three genes in blue that any animal retrovirus would have the genes that make the viral structural proteins gag, pole, and... But as you can see in different cultures it's divided by the light but I don't know if it's red and yellow or whatever but those that are not in blue are the extra regulatory genes. Some of them parallel almost exactly what we know for the leukemia viruses. Others as I said earlier we do not yet know enough about their function either in the replication cycle or in avoiding the immune response to them. Now let's look at the disease and the remaining part of my talk and in directions we're trying to go through something about this virus. There are... AIDS is called a syndrome acquired immune deficiency syndrome for a very good reason. It's not one specific disease entity but because of the change in the immune system there are a lot of different visible manifestations and they can vary from individual to individual and in early years cause considerable confusion. I like to think of them divided into four parts. The first is the impairment of the immune system with all the problems of opportunistic infection which means microbes that wouldn't cause disease in a healthy person certain ones can do so in people with AIDS. Infection of the brain a point that for example many of us thinking about gene therapy like Dr. Glaze will talk tomorrow in our own laboratory partly in collaboration with him we have a problem even if we could have wonderful gene therapy for cells of the bone marrow could we ever protect against infection of the brain or reverse what happens in the brain? That's a complete separate pathogenesis in my mind. Probably it's the infection of the macrophage another target cell for this virus because it too has CD4 molecules on its surface like the CD4 positive T cell and gets infected and the macrophage it currently gets to the brain and you can become the source of cells there that appear to be the primary targets in the brain known as the micro wheel cell. A third aspect is an increased incidence of several types of cancers or abnormal growths for example and not listed on the slide there's a rising incidence in cancers of the genital urinary area from papilloma viruses combined with HIV there's a big increase in B cell lymphoma and there's a major increase and especially in gay men of a strange tumor that involves blood vessels and therefore appears purple called Kaposi sarcoma a point I'll come to as an example of some current research in just a moment the last point I listed is rather self-evident and it says that of course people that are HIV infected have more than the usual amount of infection not just with opportunistic agents but with real pathogens that would cause disease in most people because of risk factors and because of immune impairment things like tuberculosis and this is causing in part is one of the reasons for an increase in epidemic of tuberculosis in various parts of the world things like the leukemia viruses I just talked about which are going up in this population some of the hepatitis viruses some of the herpes viruses many other times as well when a cell is infected this can happen that's a T cell that's a normal human CD4 positive T cell that was infected by HIV if I showed you another slide which unfortunately I don't have I didn't bring with me you can have infection and see no visible change at all but when infection occurs and that T cell is activated by an immune stimulus it is programmed to die we do not know the precise mechanism of killing of the T cell by the AIDS virus but this is an easy observation to make in the laboratory infect the cell wait, looks normal immune stimulate that cell is programmed to die we know a lot of reasons we started to talk about it in the discussion after Alipas talk about why T cells decline by the infection one is direct cell killing but there's good reason to suspect that there are many indirect ways for the T cells to decline harm to the thymus gland is one the envelope of the AIDS virus is called lycoprotein 120 GP120, 120 because that's its molecular weight 120,000 that envelope can be found free in the serum separate from the virus when there's active virus replication and that goes to a question that came up earlier in the first period of latency there is clinically but not with the virus when you get infected throughout the time of infection you can always find cells expressing the virus the virus just doesn't go into all cells and say I'm going to hide for 10 years then come out and cause disease that's not how it works you just reach thresholds I believe of immune impairment when disease becomes manifest but all through this problem there's virus replication you just have to handle it and bring it down a bit and then variants of the virus come out and those variants are escaping the immune system that's already responded and by this time there's enough T cells knocked off and enough impairment that there's even more replication as disease becomes manifest it's easier to isolate virus and so on and you have more rapidly replicating viruses come out GP120, the envelope of the virus in a few days we could talk about it and perhaps even have a mini symposium from laboratory experiments of how they may make a T cell number decline without the virus ever infecting the cell in other words if you have a population of human T cells in an HIV infected person only a small number of those T cells are infected but the majority are impaired in their ability to respond to an antigen and the majority are impaired in their ability to produce a T cell called interleukin-2 and some of this it appears involves complex interactions with free envelope that's floating around most recently my friend in Paris Daniels agree and his colleagues have made a very exciting discovery it's not yet published but they have found that there's a sequence of the envelope of GP120 the AIDS virus envelope the outer code it's identical to sequences of the molecule that is its target CD4 molecule it turns out that that sequence of identity is critical in laboratory experiments for the ability of that T cell to respond properly to foreign antigens which is a hallmark of the dysfunction seen in HIV infected people I believe that story is going to unfold in a very important way during this coming year so we're learning from these laboratory experiments and we're trying to see what's the most important of these experiments for the in vivo phenomenon now this is one of the great ways the virus escapes from the immune system and this is viral variation this, as you know, the AIDS virus when it's isolated from any two people is different it may vary 2%, 5%, 10%, 15%, 20% what is often not appreciated is that in any one individual there are minor variants but the minor variants have profound biological significance this slide is a cartoon that illustrates one type of virus in one individual and at one particular period of time let us say the color blue dominates you develop an immune response here listed as neutralizing antibody which is only one type of immune response that you make against the virus the other is the cellular arm that you heard about this morning that neutralizing antibody may take care of what you see in blue but in time red or purple whatever it is I can't see it may emerge it may not deal with that and if that virus then takes hold in other words the immune system is constantly running after its tail there are multiple ways that we know this slide is far from complete about how HIV escapes from the immune system the first is important integration and latency at a cellular level not at a total clinical level in other words in any one cell you may have integration and quiet no virus expression immune system can't see it the second one is let's forget number two the infection of the brain where the immune system can't do all of its work the third point is the steady but insidious destruction of the immune system the fourth point is the variation in the virus now I'd like to conclude with a brief discussion of the neoplasis associated with HIV infection I'm going to mention only the P cell lymphoma and Kaposi sarcoma and the P cell lymphoma I'll mention just briefly the tremendous epidemic now in HIV infected people of P cell lymphoma I do not know the precise statistics I've heard that it's as much as about one in three infected people if they live ten years will get this particular cancer now it is interesting the virus HIV is not a tumor virus it's not a transforming retrovirus it's not like HTLV1 the leukemia virus when you look at the tumor the role of HIV must be indirect it's affecting a cell here but the tumor appears here what clues we have are the following infection by HIV with all of its proteins chronically present over a long period of time are driving proliferation of P cells polyactivation of P cells some of this leads to an abnormal increased level in the serum of certain cytokines and also Dr. Marek referred to at some points in your talk those cytokines are released by activated cells of certain types and in particular cells of the immune system and their function is to carry intercellular messages one cell telling another cell grow, stop growing, differentiate do this, do that, it's usually in response to an inflammation or wound healing or in an immune response to a foreign antigen in HIV infection we have an increase in the presence of certain cytokines some of those cytokines may be pushing growth, proliferation of P cells and then you have the chance of an abnormal chromosome rearrangement that is known to occur before these lymphomas take place just an accident because of over proliferation chance and I'd like to turn to a little more discussion of this strange tumor Capacy sarcoma Capacy sarcoma has existed for a long time long before the HIV epidemic Capacy sarcoma can occur without HIV again the tumor that we study does not have HIV in the tumor cells HIV is not making that a tumor directly once again if HIV plays a role it's indirect HIV is here the tumor is here Capacy sarcoma appears to be increased in any population of HIV infected people but what's interesting is more much more in gay men why? how does HIV do this these are the interesting questions another interesting question around for a long time is Capacy sarcoma really a cancer when you see lesions like on the surface of this man's back lesion in one spot lesion in another did one feed the other or do each of these arise de novo is it real cancer with metastasis or is the internal milieu of the individual abnormal and at certain sites we have abnormal growth of these blood vessels and other cells infiltrating as we see in this tumor that's a very interesting question Capacy can go on to become much worse as shown in this man and can produce obstruction of the lymphatic system and can release its cells can release cytokines that make vascular capillaries more permeable so you can get tremendous edema leading to a lot of suffering by people who have this tumor as shown in this man's face now because of the increase in gay men many people thought there might be still another microbe yet to be discovered in Capacy about five years ago we set out to try to find if we could confirm that or reject it we have never found evidence for any other for any microbe associated specifically with Capacy sarcoma but what we learned was the following we developed some techniques for culturing I think for the first time what we believe are the Capacy sarcoma real tumor cells those cells are spindle shaped when you look at a tumor and most people believe most pathologists believe that they come from some cell of the walls of blood vessels the inner lining is called endothelium of course and it's some endothelial cell or maybe a progenitor endothelium that becomes Capacy sarcoma when you look at Capacy sarcoma histologically to a primitive pathologist that's me I don't look at slides very often to me it looks like wound healing wound healing that doesn't know how to stop lots of blood vessels, lots of infiltrating cells lots of edema it's as if you scrape your hand and you take a piece of that study it under the microscope but it doesn't know how to stop when we cultured cells which are shown in the next slide to make a long story short this is back to 1988 to 90 we found that those cells are producing factors that make blood vessels grow in other words the dominant spindle cell of a Capacy tumor produces cytokines that make blood vessels grow we put these cells into a nude mouse a mouse that doesn't have thymus gland function and what we expected was that the human tumor cells would grow in the mouse what we found is that those cells are not human but mouse when we evaluated them histologically they look like early Capacy sarcoma those circular things with red little balls in them are newly formed blood vessels so the mouse develops a reactive phenomena to the cells we inoculated and develops a mouse KS like lesion but it only survives as long as the human cells in the mouse survive the human Capacy sarcomas don't behave like a real malignancy a real malignancy will usually grow as a human tumor in the mouse so instead of using the cells we use the fluid that the cells are bathed in do the cells secret something that makes the tumor and the answer is yes it's not a virus it's cytokines so when we put the fluid into the mouse you develop a tumor now what are these cytokines what are these molecules that these cells are making this is a bad slide for a general audience don't bother looking at it it's just my reminder the things that are listed there are what we call cytokines and plus and minus means that these endothelial like spindle cells from patients with Capacy sarcoma are making large amounts of certain cytokines among them I want to tell you that we believe the most important is the one at the top B FGF FGF is fibroblast growth factor B just means basic that molecule is one of the most potent molecules for making blood vessels form that scientists know about for many reasons we think it's critical to the sustained growth of these cells and for making of the blood vessels let me kind of reach the last point on Capacy's that I want to deal with and that is why does HIV lead to such a big increase our hints are two fold number one I told you earlier that infection of the immune system by HIV somehow leads to an increased making and release of certain cytokines not FGF not the BFGF I just mentioned but things with other names of other cytokines those cytokines have the effect of activating endothelial cells and also making them proliferate once those cells are activated and proliferating they're making their own cytokines like the BFGF I told you that are even more efficient drivers of their own proliferation and of forming new blood vessels HIV has something else special we found some years ago and we is principally postdoctoral in my lab now becoming a senior scientist Barbara Hensley that one of the HIV special regulatory genes called TAT in some respects the analog of what I told you about in the leukemia virus TAT was known to be an extra gene in HIV necessary to its replication but what we have found is that TAT can be excreted by HIV infected cells in the early phase of infection and when it's excreted it's taken up by these spindle cells as probably poorly shown in this slide because I can't see it at all I don't know if you can but those spindle cells can take in an extra cellular TAT that is secreted by an HIV infected cell and the point of all this is that TAT is a growth factor also for these spindle cells so we think we've learned two ways that HIV may promote capicy sarcoma and in both of them it's not immune deficiency it's immune stimulation and in my view AIDS is a misnomer yes it's acquired yes it's a syndrome but it's immune but it's not just deficiency it's also abnormal stimulation it's abnormal making and release of cytokines that do stimulation not deficiency to me capicy sarcoma is arising in much greater incidence with HIV because of this provoked immune stimulation these cytokines it's set a process that becomes somewhat relentless a final slide on TAT is not yet published but we can show that this molecule will make normal endothelium form tubules like blood vessels this in a large number of other studies with this molecule not yet published indicate to us that TAT is mimicking in many many respects a normal blood vessel forming factor in other words mimics something like the basic fibroblast growth factor it makes the normal endothelium line up like a tubule it makes the cell activated it makes it grow it does other things that I don't have time to tell you about now let me summarize with this two schematic slides I've told you shown at the top that a retrovirus can be involved in the formation of a cancer by a direct transforming effect HDLV1 for example can infect its target cell shown at the top as cell A that cell can become immortalized but it may be controlled by the immune system or by other parameters but if another genetic accident or two occurs from hyper proliferation then you have a tumor and every cell of the tumor will have the viral genes in it at the same place listed in that as X so they're the descendants of a single transformed cell, clonal like a true cancer but I've also told you with B cell lymphoma of man in Kaposi's sarcoma a retrovirus namely HIV indirectly where it affects a cell let's call it A which would be cells of the immune system but the cell that becomes a tumor is much different and it's a complex process that is summarized very broadly in this slide HIV infection of the immune system one of the features is that there will be an abdurant release of certain cytokines that's far left some of those cytokines will lead to activation of cells that are the progenitor cells perhaps or even a mature endothelial cell cells that line blood vessels when those cells are proliferating activated we believe you'll have Kaposi's sarcoma like lesions those lesions I believe therefore are not a true malignancy but what we call a hyperplasia and by this kind of argument this tumor should be reversible and rarely one does see Kaposi's sarcoma reversible but instead I must make the important caveat that there are patients especially in late states Kaposi where these lesions apparently at least by pathology eyes and by clinical response convert to full scale malignancies sarcomas but that could be because of hyper proliferation over a long period of time with increased risk for other genetic changes in a final minute I'd like to tell you what we want to do about this disease in the laboratory point of view right now the only real practical advance we have in AIDS is the monitoring system and the blood transfusion protection with the antibody test all of you are aware also however of an advance made some years ago particularly by Dr. Broderen colleagues at NCI in the development of AZT which grows well AZT helps but it's far from the ultimate answer remember that we face unusual challenges people always want to know why you make so much progress in the early years etiology and blood tests lots of molecular biology advances but you can't cure the disease remember it's an integrating virus that a virus usurps the cells machinery that we have to develop much finer tools than we do against the bacterium or fungus because it has no metabolism in that cell it's using the cells enzymes for the most part so we have limited targets that are specific to the virus and they have to be very sharp to do something to the virus without a host of toxicity and remember if it integrates once it integrates and you knock out viruses all over the place once you stop treatment virus could stop emerging start emerging again and then remember how fast this virus mutates it mutates away from the efficacy with AZT talking about a vaccine why so much trouble with a vaccine to my knowledge nobody has yet developed a vaccine that prevents against infection Jonas Salf Salf the polio vaccine as far as I know doesn't protect you or me from getting infected protects against disease we're vaccinated but if we get exposed to polio as my understanding is we'll have replication of that virus in our gut but it won't get to our brain in spinal cord because the immune system is activated and controls it but imagine a retrovirus which if you don't protect against infection any virus that's integrated the immune system the immune system in a vaccine may protect against infection but if you escape and integrate the immune system holds it in check as the immune system declines if you're not boosted regularly the virus will come out maybe see the brain again so we have argued that we need to develop a vaccine that protects not against disease but against actual infection and that's been the goal second thing to keep in mind is there's never been success in a vaccine we have a lot of variation look at influenza, we have an imperfect vaccine and influenza is a child compared to this virus out of AIDS if there's success with a vaccine one might safely predict we ought to have a vaccine against anything if science puts their mind to it enough my own view is that it's not so much the total money but maybe we need to have a strategy that encompasses a non-secret mini Manhattan project for this disease and this virus I fought this for a while it sounds almost like a cliche but if you think about it deeper there are some good ideas that might emerge I'm going to close by showing you an example of what we're trying to do in collaborations with many people I'm focusing on our laboratory is focusing on two approaches in therapy for the future one is gene therapy which Michael talked about tomorrow and I'll just mention it very very quickly and the other is something we call anti-sense many of you may heard about anti-sense can be used as a drug or in gene therapy and it means if you know a sequence let's say of the virus you know it's gene sequence and you know it's important you can build what is called the anti-sense that binds to it and it blocks that virus nucleic acid from acting as a messenger to make viral proteins actually it may do much more than that but that's the common common way it's discussed so our program is listed here with Mike and other people at NIH and elsewhere in some aspects of gene therapy one thing we want to do let's just look at the top one is block TAP that molecule that's critical for viral replication and is also we believe a growth factor for capacy and probably doing many other bad things for TAP to function it has to interact actually indirectly it's very complicated but with a sequence that the virus has in its genetic information called TAR our notion is that if we put into a cell a protective element genetic sequence we may protect that cell from ever being infected and the notion using the binding site for TAP is summarized in this slide on the top we see a cell that's infected carrying the DNA form of the virus integrated and it's making it's going to make its TAP but if we insert into that cell ahead of time a protective gene that has binding sites called TAR many times as soon as TAP is made it's swamped up by the TARS that are made by the protective genetic element you might argue but that might not be good for you to put poly TAR into a cell but it's only a cell that's going to be infected that's going to be activated and that's how this is worked out so any cell that gets infected poly TAR is expressed and gobbles up and binds to the TAP so TAP cannot work and the hope is to do this with stem cells in the near future and that's one of the ongoing collaborations lastly I'll give you an example with antisense this shows ribosome messenger RNA let's say that's a viral messenger RNA being translated to make a viral protein but an antisense DNA sequence a relatively short fragment let's say 25 nucleotides combine to that and protect it from being translated and very specific and you can do it as gene therapy in the DNA form or you can do it as a drug that would need to be around chronically I'll give you one last example with data in two slides I told you that one of the important molecules for capacy sarcoma in our minds is the cytokine called basic fibroblast growth factor look in the middle basic fibroblast growth factor is over expressed in capacy spindle cells basic fibroblast growth factor is released in an active form and acts to promote growth of those spindle cells in vivo people have seen basic fibroblast growth factor expressed a lot in capacy sarcoma so we are using some antisense approach this is my last slide it's a data slide all you need to look at is the right half and look in black and that's the effect of a specific antisense in blocking growth evades capacy sarcoma cells in vitro these in vitro ideas we hope to take in vivo to the clinic in the immediate future I believe immune therapy and immune restoration has a function in the future of AIDS research we're not so involved in it but I think it's a promising avenue and then I think these next few years are going to see more and more of that the protective vaccine in my closing conclusion remark is not going to come in the next five years one can predict that several vaccines will emerge some might be partially effective but one would not want to state if and when the vaccine will come within this decade thank you very much