 This is the second time that I've had the privilege and the honor of introducing Dr. Robert Gallo to a Nobel conference. When he was here six years ago for the conference on immunology, he heralded from the prestigious National Institutes of Health in Bethesda, Maryland, where he'd served in several positions culminating as Chief of the Laboratory of Tumor Cell Biology, a position he'd held since 1972. During his time at NIH, Dr. Gallo was distinguished by bold innovation, discovery, and accomplishment. He discovered interleukin-2, which is a substance which allowed scientists to grow T-cells for the first time. He then isolated the human T-cell leukemia virus, HTLV1, which was the first human retrovirus to be discovered after painstaking search by others. This discovery earning him the title Father of Human Retrovirology. He authored and coauthored around a thousand articles and books. In many awards, he was chosen to receive the Albert Lasker Award in Medicine twice, which is unprecedented. He identified the virus responsible for the mysterious and horrifying new disease, AIDS, and from there he developed the AIDS antibody test used to screen and protect the safety of the nation's blood supply. He was also the most referenced scientist in the world during the 1980s. Three years ago, Dr. Gallo left NIH to head up an entirely new institute, the Institute of Human Virology, or IHP as it's called, housed in the Medical Biotechnology Center at the University of Maryland. Dr. Gallo's new institute is unique in its multidisciplinary approach to the study of disease, combining basic research, epidemiology and preventive medicine, vaccine research, and clinical research, which is a remarkable combination, toward the discovery of diagnostics, therapeutics, and vaccines in human virology. There is much hope and expectation in the medical community that this institute, under Dr. Gallo's leadership, will be the locus of the next big step in the struggle against AIDS, an effective, affordable treatment. This will, of course, be an incalculable historical contribution towards the well-being of humankind. Toward that next beachhead, Dr. Gallo and his colleagues recently discovered that certain naturally occurring substances called chemokines are strong inhibitors of HIV infection. This discovery helped lead to further discovery about the mechanisms whereby HIV enters cells. Science magazine honored this discovery in combination with the pharmaceutical industry's development of the protease inhibitors as the most important scientific breakthrough in 1996. Dr. Gallo counts among the innumerable early influences on his life, a hard-working father, a pathologist friend who thrilled him with medical detective stories, and a zoologist uncle who found fun in his professional life. It doesn't take a long conversation with Dr. Gallo to realize that this hard-working master investigator of medical mysteries is having fun. His colleagues at HVI know it, and they find his lively attitude toward work infectious. As one of them was quoted recently saying, the air crackles when he's here. Well, he's not there today. He's here. So once you please welcome Dr. Robert Gallo. I want to begin by, well, thank you very, very much for those kind remarks. I also want to thank Dr. Hilbert for being my shepherd these past day and a half. I have not had to think for one second until I think I have to think now. And I also want to thank Neil. Pronunciation is going to be a little difficult. Woodensack, I did it, for being, well, he's the other half, the shepherd and the guardian angel. Thanks to the college for inviting me back, and I think all of us have a feeling that there had to be a lot of fortitude and perseverance to do what you did from the time of the great tornado here. I guess there also had to be some faith, hope, and charity involved in all of that. I'm going to make the talk that I give a little more overview-ish than the title says, at least with a little more background, I think it's appropriate. And I hope the talk can maintain the wonderful quality of the talks yesterday, who certainly make this job easier and more pleasant. Let's begin by looking at a picture of HIV, just to get an idea of what retroviruses look like. This slide shows not a cell being infected by HIV, but shows a cell that's already been infected and is now producing the virus. So some patient electron microscopist did this. I would never have that patience, because in order to find the various steps in the release of the virus, what we call budding and pinching off requires looking at a great deal of pictures. But in any case, that's what happens with several kinds of viruses, retroviruses, a classic example of budding and pinching off from the cell surfaces as the virus forms. As you can see, I think in the upper left corner of this slide, the diameter of a retrovirus is about 1,000 angstroms, 100 nanometers. You can see something dark in the middle that's called an electron-dense substance. That's where the virus's genetic material resides. You heard yesterday from Dr. Jocklick and Dr. Holland about RNA viruses a great deal in a rather sophisticated manner. You remember that RNA viruses have a lot of variation, HIV being a retrovirus is also an RNA virus, as I think you heard also yesterday. So it has lots of variation, as you know, can undergo lots of mutations and exists in an individual as swarms as was described yesterday. What's unique about a retrovirus for an RNA virus is that once it infects a cell, soon thereafter it can essentially convert its genetic information from RNA into DNA. In fact, this really gives it its name. Even more importantly, or part of this, and really the essence of a retrovirus is that that DNA integrates, like being sewed in to our chromosomal DNA. So we picture it now, once a cell gets infected, the viral genetic information is like our genes, it's in our DNA, and so therefore the cell, unless it dies, is infected forever more. But if the cell divides, the viral genes are transmitted to the daughter cells, the progeny cells, so that in general infection is for the life of the organism, animal or human depending on the retrovirus. That's really the sine qua non of any retrovirus. DNA formation from RNA, integration, permanent infection. Usually in animal models, and even before AIDS when we thought of human retroviruses, we tended to think of them in causing some kinds of cancer. Many in laboratory experiments, but often also in nature. Usually leukemias or lymphomas, sometimes sarcomas, occasionally a carcinoma like a breast cancer. So they were really the subject of the National Cancer Institute and mainly cancer-orientated virologists, and then also for some rather more pure molecular virologists. But AIDS dramatically changed that, but in retrospect we knew that among retroviruses of animals, they could cause also non-malignant diseases, including immune deficiency. You probably know that the major target cells for HIV are cells that are critical to the immune system, certain types of cells that are called T cells and generally certain subsets of T cells called CD4 positive T cells. So they are the principal target, but also another important cell in immunology, antigen presenting cells, particularly a cell called the macrophage and elsewhere cells called dendritic cells can be targets for HIV. And I'm going to spend considerable time on the mechanism of how the virus gets into the cell because it's new and we think important. I believe there are three major areas. I think most people in the field would say this, I'm rather obvious. Three major issues that we're dealing with in HIV research right now. One is the development of better therapy, constantly needing improvement. And the second is the development of preventive vaccine, not in any order of importance. We have to have a preventive vaccine. And a third could be debatable as being in the top three, but I think merits it. Is the reason or reasons I should say why some people resist infection, that is to say some people are virtually uninfectable by HIV. Something we've only learned in recent years. And there are other people who do get infected, but who progress much slower compared to other people. What are the reasons for that? We think learning those reasons will be very important to the development of vaccine and for further better therapy. We can in fact say that research on this matter was stimulated more by patients than by scientists. People within the gay community kept bringing out the fact that it seemed that some people were not infectable. And also that some people without therapy were doing rather fine, with only minimal involvement to their immune system. But now we're beginning to get a handle on it. Before addressing in more detail these three things, let's talk a little bit about the epidemic and about the origin of the virus. Like life in general, I think we can now say somewhat conflict here. We can say with assurance that HIV originated in Africa. We say that because some of the oldest positive cases can be traced back to Africa and also because there's a widespread among African subhuman primates of infections by related viruses that are known as a simian immunodeficiency virus of SIV. Some of them being extremely closely related to some of the viruses in us. It seems obvious that monkeys would have infected men for long periods of time. I cannot believe a recent infection of humans by this virus. Rather, I think it's the epidemic that is recent. One could argue that maybe it's a recent mutation of older viruses adapted to humans, maybe so. But it's very likely that within the monkey populations, viruses infected humans over the period of time, many times, because humans eat monkeys and keep them for pets. You have to skin them, hunters get cut, blood entry is obvious, would be recurrent and for long periods of time. But I think probably if this did occur, as I suspect anyway, people died with their disease. It didn't cluster in an epidemic form and you heard important discussions of spreads of epidemic yesterday from Dr. Crosby. So we can think about a new viral disease, at least in these three ways, that it's a mutation of an old virus that was already in humans. Or you can say it came into humans relatively recently from monkeys, or animals of some sort, or at least its origin, rather, was among animals, zoonosis number two. And then we can say also that maybe it was in humans, but there had been a sudden transmission to a wider population. In a sense, maybe all three applied to HIV. Certainly it came into humans from monkeys. Certainly it exploded relatively recently, sometime probably in the 60s, early 70s, late 60s, mid 60s, and maybe, maybe there was mutations of a number of these primate viruses that entered humans until some of the better ones that could adapt to humans took hold. What accounted for the sudden emergence? You could guess it if you don't know already from talks yesterday. This was a virus of the rainforest. When colonial powers left Africa, there were the development of some local wars, famine, migration of people. I've heard it said by sociologists, historians, that some of the people lost the old ways and didn't learn well enough the new ways, so there was big migration into cities. Think of Kinshasa going from logarithmically increasing over a decade in population, fostering a big increase in prostitution. So you might say the rainforest came to the city. But how did the rainforest come to the world? That happened relatively fast. And the best guess, of course, again, as you heard yesterday, airplane and travel, we could add to that an increase in sexual promiscuity during part of that period. You remember, if you go back far enough, before AIDS, there was these herpes simplex two epidemics and other epidemics being discussed due to increased promiscuity. But also blood and blood products were being moved from nation to nation. It wasn't true, I think, pre-World War II. And also the insane international habit that developed of intravenous drug abuse. All these things could make a blood-borne or sexually-borne virus much more transmissible so that the rainforest became global. So something remote, something rare, became something relatively common and something global. Another reason we think of Africa as the origin other than the monkeys, other than the old cases or serum samples earlier than elsewhere, linking it to Africa, is the fact that the greatest variation of the virus can be found in Africa. I'm sorry, I must have bypassed the slide. Yes, here. I don't know if this shows up, but if it does show, and you are not colorblind, you can get the point from looking quickly at this slide. Yesterday, we heard in that beautiful overview by Dr. Holland about the great variation and the great mutations and the swarms of RNA-type of viruses that exist within a population. I've got to boil this down into biology and clinical medicine now, and I'm gonna have to take some liberties and go as far as I can. I can't go beyond because I don't understand it beyond that, and it'll get more complicated for me, let alone for you. But we can say that, first of all, when HIV was first found, we began to see that viruses in the United States from person to person were different. They were never exactly the same. And then we found and published that, within an individual, what you heard from Dr. Holland yesterday, there were swarms. We called them microvariants. So even though two different people would be infected in the same group, both would have very significantly enough different HIV ones, and within the individual, there would be these microvariants or these swarms evolving. Now, in addition to that, there are what we might call commonly strains of the virus. Better is a genetic term called clade, C-L-A-D-E. In the United States, we have only clade B. That means that although the virus differs within us, and from individual to individual, all the viruses in the United States can be lumped together within a certain genetic variation we call B. But there's also A, C, D, E, F, G, et cetera, and they're all in Africa. And from Africa to Asia, we now see other of these clades emerging, have seen it for several years. Max Essex and his colleagues believe, but it's not yet well substantiated or documented, that some of those clades are more readily heterosexually transmitted. A great public health question that needs very careful attention. I think he thinks clade C, and or D, in parts of India, Thailand, Botswana, are more readily transmitted heterosexually. And he argues that that may be one of the main reasons for the dramatic rise in Zimbabwe and Botswana in the last few years and in South Africa. In any case, the purpose of the slide was to show you that there's more color in Africa than elsewhere, greater variation, greater length of time for evolution of the virus. That is, it was there first. Now, since I was here in 1992, there was a period, let's say four, five, maybe up to 1996, where there was periodic good news being reported in the newspapers and elsewhere. One was related to the epidemic that it had plateaued or stabilized. Another was related to therapy and the development for the first time of some practical advances other than the blood test that is reasonably good therapy for HIV-infected people. The first of these, the epidemic, is only a little bit true and only true in some special context. The second is true, but still problematic. I'm gonna speak to both of them now. Regarding the epidemic, yes, it has plateaued in some parts of the world and in Umchung populations, but how about in black Americans? You can look in this slide and see young black men and deaths from AIDS. But let's talk about, not people already with AIDS, how about infection? In parts of Baltimore where I work, as many as 10% of some neighborhoods are HIV positive, most in the minority black populations. It's a rising, frightening epidemic in the black community. In fact, as you probably know, Fumi and Julian Bond have meeting with the vice president and others have now made the National Association for the Advancement of Colored People's major highlight or problem no longer violence being number one, but AIDS. And we have been talking to both about things we might be able to do within some communities in America. You may think that everybody in America has the so-called triple drug therapy and everybody's doing fine, but that's not true. Not only is not everybody doing fine, a point I'll come back to, but not everybody gets the therapy and it's not always the problem for the, or the fault of the health people or the doctors or the hospitals. There are many people in the black community in the United States that don't trust and don't come in and reaching them is going to be a serious problem, is a serious problem. Now, we think that the best way to have this improve is through the churches in the black community, but also those of us in the medical community interacting more and taking more black students in all walks of life, meaning like high school, college and so on, all the way up in various, even short term for a few weeks into laboratories because they can bring messages back more readily than some of us can. In any case, there are black populations in the United States that are receiving virtually no therapy, not virtually, no therapy or poor therapy and are not adequately followed and the number is not small. Bob Redfield, the head of our clinical division at IHV, tripled the patient population right in our region in about a year and a half. We've been operational for two years in roughly a year and a half, he's tripled the patient population just by going out and trying to make contacts with community so there are many more infected people and many more people actually with AIDS than we even knew when we arrived. Okay, let's look at one last epidemiology slide. This won't project, well, I can tell from yesterday looking at the slides, but again, you can look at colors. I think the bottom are AIDS cases and the top is infection and the different colors are different parts of the world. Red is Africa, if I remember right, I can't read this at all. And I think yellow is Southern Asia. You can see plateaus in Europe and the United States in some of those other colors but just look at the situation in Africa. Oh, I have a pointer, I'm sorry. You can say it's plateaued but look at the level of plateau, that's certainly not good news. What would good news be? You can't say that plateauing with an enormous rate of infection is good, it can't go much higher. Some people say that there are villages in Africa or towns where the number one population is orphans, are orphans because of the death of course of the parents from HIV infection. Now, I said that there were really advances particularly in therapy. And now let's look at where these advances in therapy occurred developed by the pharmaceutical industries in the last few years. Of course it was based on basic information of the replication cycle of HIV but frankly, we knew this, a good part of this for almost any retrovirus. There are nuances for HIV but they haven't yet been taken advantage of in therapy. If this is a cell, this big rectangle, that's the nucleus of the cell, we can look at virus infection as incoming and let's call it outgoing or formation of the virus particles. Like any retrovirus, HIV has to find something on the surface of the cell to interact with. It's important for the rest of my talk to know something about a cell. If I had my fist representing a cell, let's say that each of my knuckles was something on the surface that's there to look for signals in the environment to know how to behave. We call them receptors and they're complicated, they're more than one kind and they do important biochemical signaling to the cell nucleus and a variety of other things. Many times, sometimes they don't signal the nucleus, they do something else. Now some cells will have all the knuckles expressed, some will have some, some will have others which means some of the cells will be able to respond to this or that signal but not another and it becomes imperative to understand that. This slide was made in 1988. At that time, we knew that HIV bound to a cell surface molecule on T cells and macrophage called CD4 and the name's not important and that's particularly why we believe it targeted those cells. We now have recent and very important information that the story is more complex, far more important and far more available to us to take advantage of. And I'm going to talk about that in the meat of this talk, I hope, as one of the major mechanisms of genetic resistance of HIV infection or progression. It'll be at this level. But let's go back to this 1988 slide. Following binding to a molecule on the cell surface, there is a fatty bilayer, lipid substance within the virus, just internal to these knobs. The knobs are what we call the viral envelope. It is a sugar protein, therefore a glycoprotein. We name it according to its molecular size. Just internal to it and in fact, part of it is varied within a lipid bilayer. When there's proper interaction of virus with cell membrane, a fusion occurs between this fatty substance and the fatty lipid bilayer of the cell membrane. When the fusion occurs, then the guts of the virus, the core that you saw as this black stuff on the first slide are emptied into the cell side of plasm. And quickly, if the cell is in a proper metabolic state, the RNA is what we call transcribed into DNA catalyzed by the famous enzyme reverse transcriptase, a major target of the pharmaceutical industry, that enzyme. That enzyme is a DNA making enzyme. We call them DNA polymerases. We have them, but we don't have one quite like this one. So you can get a window of selectivity with drugs that target that enzyme. Later, the DNA form of the virus, much more complicated than shown in this slide. We now know much more about this. Gets to the nucleus where it integrates. Remember, we said that's the real essence of a retrovirus. So here you have an RNA virus with all its variation. That's bad. Or it sometimes can be bad for the virus, as Dr. Holland mentioned yesterday, but often can be the reason the virus escapes or revolves into more potency. In the same time, you have a virus that once infection occurs, attacks the central cells of the immune system. And thirdly, you have it converted in a way into a DNA virus for a while so that it persists. And it can be silently persisting without seeing the virus again just in the DNA form, escaping the immune system. But if the cell is in a proper metabolic state, the DNA is expressed, requiring some viral proteins to be made first, known as TAT, TAT and REV. I'm gonna speak again about TAT. Therefore, I mentioned them now. I won't go through the processes of how they work, but they're necessary for viral RNA to mature, to get out of the nucleus into the cytoplasm, and for the messenger RNAs of the virus to begin to make viral proteins. They make viral proteins sometimes in a very big size, which have to be cut. The viral proteins are cut by enzymes that are called proteases. That happens from a cellular protease and from an HIV-specific protease, another target for the pharmaceutical industry is the HIV protease. Therefore, you have the term protease inhibitors. When you have the proper viral proteins in genomic RNA, they assemble at the cell membrane and with a complex series of steps, you have the pinching and the formation of the virus as I showed you on the first slide. Okay, now I told you that the pharmaceutical industry concentrates on these two enzymes. Uh-oh. Blank. Something happened. Um, that's one behind, so I got one back. Yeah, that's it. Actually, this is unneeded, isn't it? We already said it. The drugs today target these two enzymes, one in three. People have been trying to develop something against number two, but not successfully so far. Well, what's the situation with the antiviral drugs today? Generally, a combination of three are used. They are potent inhibitors of HIV. They result in a tremendous drop in virus in most treated people, and they essentially have converted a significant percentage of HIV-infected people from dying within seven, eight, nine, 10, 11 years to a chronic disease that many people can live with properly managed, closely being followed by physicians generally at a medical center. Now, does that mean the problem is over? With these dramatic results coming out in 1995 and especially in 1996, some scientists in the field argued that we could turn most of our attention to the development of a preventive vaccine. We argued then and would argue much more strongly now that this is a little dangerous. Though we have to develop a preventive vaccine, we can't do so by lessening research on developing better therapy. For a number of reasons, some of which are listed on the next slide. Well, no, the next slide actually shows people on triple drug therapy doing much better than people without, just an example. But what are some of the problems? Let's concentrate on number two. There is insufficient time to judge escape mutations. In fact, we're seeing escape mutations from these drugs now. In addition, there can be significant toxicity from some of the drugs. The hope was that if virus could be held down for one or two years based on some mathematical models that were around during 1995, 1996, that even if the virus was present in some other cells, if you blocked virus spread, the cells harboring the viral genes would die of old age and you might cure the disease. That proved to be an assumption without basis because the cells that carry virus live much longer than people knew or know now. In fact, we don't know the lifespan of many of the target cells. And there continues to be some cells that are producing low levels of virus constantly that can be demonstrated for the vast majority of people, even under the best of therapy. Therefore it means long-term treatment. Those of us who saw early cancer chemotherapy go through many years, even almost decades, before it could be worked out even in one disease, childhood leukemia, realize that if it's for years, we're gonna get toxicity and that is occurring. We have problems also with compliance and of course with cost. And last, but not least, what I mentioned already, reaching enough of some populations in the United States, for example, but also 90% of HIV-infected people or more worldwide receive no therapy. One could argue that this is an economic and moral problem, right? When you think about it a little more, it's also a logistic, impossible problem. If you had all the money on earth, could you solve the logistics of following all these patients in rural regions, let's say, of equatorial Africa? So the problem is not just money for drugs, it's logistics, because this kind of therapy requires careful follow-up people, especially at the beginning, even doing viral titers, et cetera. Therefore, we have argued that it is important, even while trying to develop a preventive vaccine, not to lessen basic research on the biology of the virus, basic studies on how the virus causes disease, how, what are the fine points of its replication cycle? Because out of such research, we think that new therapies could be developed, hopefully more biological ones, with less toxicity and less possibilities for resistance and hopefully some of them might be able to reach the third world. For the remaining part of this talk, I'd like to describe to you three areas of research involved in our institute, one of which is extremely collaborative with groups in Europe that I'll refer to in a moment. They don't answer the prayers, but at least one of them has a good chance of providing therapy in the third world, at least in my mind, and I believe that's going to happen in 1999 or at least by the year 2000 to some extent. But that'll be the second thing I describe time permitting. Well, let's go back now to, well, this is the title I want to make for the rest of the talk. Three approaches of biological control of HIV, let's say, semi-natural approaches. And I will start with going back to this slide, to the beginning of virus infection. I mentioned to you that this slide was made in 1988, so it's 10 years old and outdated. The pathway is fine. However, there's much more complexity, especially to the first stages of virus infection. And it is that I want to now go into in some depth with you. This could be the more difficult part of the talk. Yesterday, John Holland said, pay attention. I think it's not so hard, what I have to say, but sometimes new terms and I forget to define them. So I want to go through this fairly carefully. We now know that there's much more than the CD4 molecule involved. Discoveries recently from a few laboratories, including work that we are involved in, have led to an appreciation that sugar molecules are important. And that's the least studied thing right now in the field. I'm only going to come back to it once or twice, because it's, as I said, least studied. But they're important. They're molecules that just sit on the cell surface, don't penetrate the membrane. They're called glycosaminoglycans. But in addition, we know that another surface receptor is actually the doorway by which HIV enters the cell. Now this work was referred to in the rather generous introduction by Richard. It was what Science Magazine was referring to about chemokines and their receptors, chemokine blockade of HIV infection. The story goes back to the, actually goes back to the mid-1980s when some workers had described something made by our immune cells that worked in blocking HIV, maybe one, maybe 10 things they didn't identify over a period of 10 years went by. In doing vaccine research in monkeys, we were frustrated that we never knew why sometimes a vaccine worked. One animal was protected, one animal wasn't, with a candidate vaccine, and it wasn't the amount of antibody we could get a correlation with, nor the amount of what we call killer T cells per se. And we wondered, could there be something else? Is there something to the story about something soluble, released, not antibody, that may inhibit HIV and do it potently and do it fast? So we began to purify molecules released by immune-stimulated cells, and it led us to these findings, that there are naturally occurring suppressive factors, that among the most important are molecules that are known as chemokines, particularly a family called beta chemokines, and they have funny names, ranties, Mip1-alpha, Mip1-beta, I won't talk about the last one, MDC, which is a recent subject that we reported on. Oh, what are chemokines? They are a subset of cytokines. What are cytokines? They are generally small proteins that our ways, cells, communicate. Go back to my fist, the knuckles, receptors, looking for things, looking for its signals. Maybe grow, stop growing, keep quiet, talk, et cetera. Do what you're supposed to do. If you have a receptor for some particular cytokine, then that cytokine, if it's made, will react with that receptor and tell the cell to do something. Interleukin-2 tells T cells grow. Some cytokines tell cells make antibody if you're the right cell, stop growing, et cetera. Among cytokines are a special family called chemokines. They get a special name, cause there's so many of them. Bill Hazeltine in Human Genome Sciences tells me that there's about 100 genes for 100 different chemokines. That's why they have a special name. We know of 55 or so now. That means there are more to be discovered by somebody youthful in the audience. So why so many? I don't know. What do they do? Most of the time we thought they were there for inflammation only, to bring cells to the sites of inflammation, chemokinetic, chemical attracting molecule. But we now have evidence in the literature from other groups, chemokine experts, they are important for homing, moving cells from one part of the thymus gland to another. In embryology for the formation of the vascular system, they have sometimes something to do with cell death pathways. So it's a new field actually. They really are a special set of cytokines. Anyway, blindly, we did, to be honest, I barely heard of chemokines when we found that they were the powerful HIV suppressive factors. I heard of them, but that's about it. So in any event, as we purified, we identified these guys as important inhibitors of HIV. And they inhibited by blocking virus going into the cell. We knew that, but we didn't know the mechanism nor did we speculate. Within six months, occurring at a time when I was moving from NIH, Ed Berger and his colleagues at NIH gave us the mechanism. The mechanism is chemokines block because in order to work, they have to find their receptor called chemokine receptor. There are many such receptors. A few are important for HIV. To enter the doorway, HIV needs certain chemokine receptors. But if there's a doorman outside blocking the way, HIV can enter. The doorman is the chemokine, per se, listed on this slide. This paper, the work was begun in late January and we published it at the end of 95, December of 95. This is the publication for anyone terribly interested. And here's one example. Well, no, I got ahead of myself. Here is now going back to the cell surface to look at the three major players that we now know about. We knew about CD4. This is a cell in pink. And you'll notice that CD4 is penetrating the cell membrane. That is to say there's an external component and an internal component. And so we know that HIV has to interact with that or at least most of the time does. What we didn't know until very recently is it also interacts perhaps as importantly with these sugar molecules, which don't penetrate the cell. They're just stuck there like a tree in mud. Not like the trees you've planted here that have already taken roots. But now we have also this complicated structure. This is a chemokine receptor. Those of you in biochemistry or cell biology will know the term seven-trans membrane G-coupled protein receptor. The chemokine receptors belong to that general large family of receptors, but it has its own special features. It traverses the cell membrane one, two, three, four, five, it should be seven. One, two, three, four, five, six, seven, yeah. Seven times. These things are all connected to each other, okay? So, HIV now is shown as this tremendous ball. This is the envelope, which you saw as little knobs before. Now showing it hidden. Interacts with the sugar, the black, and also CD4 looking like an explanatory point. Why? It seems from our understanding now that that interaction allows a portion of the GP120 molecule, the envelope, to find the chemokine receptor. A few labs, ours included in a nature medicine paper, reported some mapping studies. We know that a very particular region is critically involved in this reaction, a critical region of the envelope of GP120. For those following the field, it's known as the V3 loop of the major part of the envelope known as glycoprotein 120, 120,000 for its molecular weight. So we begin to now get physical, structural data on these kinds of interactions. And I indicated chemokines block. Why do they block? Because they find their receptor. And interestingly enough, they find the receptor by interacting with the same sugars. Those sugars somehow lean the chemokine to the receptor. How does that block HIV? It blocks HIV physically, wouldn't it? Like that? So HIV can't get there? Yes, but more than that. When the chemokine sits shown here in gray on the chemokine receptors in pink, it also, in addition to physically blocking that cell from being virus infected, it does what scientists call down-regulate the receptor. The receptors internalize. And new receptors for a considerable period of time are not made. So it's like the cell is now born without the receptors so HIV can't infect it for a while, okay? This becomes really rather critically important. That's an example of data we publish showing that these chemokines inhibit HIV. This is the 100% and it shows concentrations of inhibition. There's only one other experiment in that paper I wanted to show you and that's shown here. Why do I show it? It's just different strains of HIV being inhibited. Because even when we published this paper, we knew something was strange. Some HIVs were not inhibited. We really didn't understand why. We knew when it worked it blocked entry, but why this didn't get inhibited was not defined. Now once Berger and Collies gave us the mechanism that HIV enters cells by using chemokine receptors, it became clear that it uses a number of HIV receptors, but it uses two predominantly. This is a complication I'll ask you to try to remember. The two most important chemokine receptors have strange names. One is called CCR5. That's the receptor that's being employed by these viruses and they're blocked because the chemokines that we found block HIV, Ranties, Mip1 Alpha, Mip1 Beta work on that particular chemokine receptor CCR5. This guy uses another receptor CXCR4. This becomes tremendously important. Why? With all that Dr. Holland told you yesterday about variation of RNA. With all that you know about HIV variable, within my body if I'm infected, between me and you if we're both infected, et cetera, within all these swarms, within these myriads of infinite varieties. Functionally, we can divide them all into two extremes, everywhere so far that this has been studied with every clade or variant of HIV. We can divide them functionally according to those that particularly favor infection of macrophage versus the T cell. It'll hit both, but better macrophage. They are less virulent. We tend to call them NSI, non-sensitial inducing, less cytopathic. And those that are more able to infect T cells, they're more T-tropic, it turns out, those that better infect the macrophage are those that use CCR5, that are those that are inhibited by Ranties, Mip1 Alpha, and Mip1 Beta, and are those that are transmitted from individual to individual, whereas the more T-tropic, more virulent viruses generally emerge later with infection. Probably the reason is that in the genital tract and in the rectum mucosa, cells that are receiving the virus principally express on their surface the chemokine receptor CCR5, less so CXCR4, so you select for the more macrophage tropic, by mutations, you start in time to develop the more T-tropic that use the other receptor, CXCR4. Taking this hint, Hans Wiegsel and his co-workers at the Karolinska Institute in collaboration with two people with me, Micky Popovic, Lorenzo Koki, but who played very minor roles was major work was done by Mary Ann Janssen in Stockholm, definitively showed the difference between inhibition by Ranties, Nipuan Alpha and Nipuan Beta on the NSI, that is on the more macrophage tropic, that is on the viruses that use more CCR5 chemokine receptor, whereas all the islets they could obtain from patients that were in late-stage disease that were more sensation-inducing, more cytopathic, more T-cell tropic were not inhibited. In addition, they showed that if you follow patients progressing to disease in black, this is the T-cell counts. As opposed to people who don't progress, shown in gray, there's a switch in those in black from using the CCR5 kind of, well, the CCR5 kind of virus to become the CXCR4 kind of virus. They concluded in this paper, primary, that is to obtain directly from patients of the macrophage tropic or transmissible type of virus are sensitive to the beta chemokines, just as I've indicated to you, whereas the other guys, the more virulent, more T-tropic, are less sensitive or resistant. A shift from the more macrophage to the more pathogenic type occurs in parallel with loss of sensitivity to the beta chemokines. Now this slide looks a little complicated. I only want you to look at the top. I can't see it, but if my memory serves me correct on the bottom. And this just recapitulates what I've told you. There are many kinds of chemokine receptors. This is a partial list. There are many chemokines. This is a partial list. This receptor is specific for those viruses that are more T-cell tropic. This stands for T-cell lines, because that's one of the ways you test it if it can infect T-cell lines that are, and this, excuse me, pardon me, the opposite. This is the more T-cell line virus tropic, and this is the more macrophage tropic. We even understand the whys of that. A region of the viral envelope, a sequence, a certain sequence is necessary to infect more of the macrophage tropic viruses, to use CCR5, in other words, and a very different amino acid sequence, just in a small range of the virus, is essential to be more T-tropic. There are other things that contribute to that, but we know part of the answer. Okay, and remember again, that the natural ligand for these guys is the ranties, mip1-alpha and mip1-beta, so it can block those kinds of viruses. Now I wanna bring up a clinical question, an epidemiologic question. Are chemokines or their receptors important in preventing infection? Remember I told you that from really the gay community, we learned this strange story that some people are virtually uninfectable or who get infected progress less slowly. We had no idea of mechanisms and quite frankly, many of us, that means me included, hardly believed this. I thought it was poor history or chance, dosage. However, we now have at least understanding in a substantial way of a major component of these uninfectables or the people that are infected but progress less rapidly, that are disease free. And it centers around the chemokine receptor system. First of all, let us talk about CCR5. Work from several laboratories, most recently and most thoroughly by Steve O'Brien at NIH, have shown that some people are absolutely almost absolutely uninfectable, minor variants of the virus can infect. And that they lack the CCR5 protein. They lack both alleles, mother and father, so they don't make any of this protein at all, yet they live normally. And that mutation, the double mutation is found in approximately 1% of Caucasians but was not found in Asians or Africans or African-Americans. So 1% of the Caucasian population is almost absolutely uninfectable. Now if you look at one allele, just that one parent absent gene, what happens? You can be infected, as I'll show you in a moment, but you progress less slowly. But who has only one gene missing? Excuse me. Who has one gene missing? What percentage? Again, nobody in Africa that's been tested. And it seems to be a North-South gradient with Scandinavians and their descendants, presumably a number here, are infectable but don't progress to AIDS very well. And you can see how coming from around 15%, 10%, 11%, you come down to Italy and Greece for 5%, et cetera. Steve O'Brien believes that this was probably an adaptive mutation that developed when Caucasians came out of Africa, perhaps in response to a great epidemic. One wonders, could it have been something like HIV? All right, now I wanna go to the chemokines. Before you look at the slide, let me introduce what I wanna bring out. We've said that if you, the knuckle is absent, you don't get infected. Well, what about if the knuckle, instead of being in large, well, let me take that back. What about instead of the knuckle being absent, you have more of the chemokine, more of the blocker. What if you and I differ in the amount of chemokines we make? Just like we do in our red blood cell count in a variety of other things. Here, the genetics is nowhere. That is, we don't have proof of genetic differences between us that would control the production of chemokines. However, we have correlative data based on determining the amount of chemokines made by you and me. And I'm gonna show you really to me a startling and amazing story that I found very unexpected in the collaboration I was involved with. It's a paper that was published in the US Proceedings of the National Academy of Science some months ago, which I collaborated in. The first author, Daniel Zaguri, is not a postdoctoral fellow. He's a senior scientist at the University of Paris. And actually, it's a multinational collaborative study. It involves a hemophilia clinic. If I can go to the previous slide, it's actually wrong, it should say 1998. A hemophilia clinic where the patients were infused with HIV-infected factor A from 1981 to 1985 before the blood test was available, who resisted infection. And I'll show you that data carefully. By the way, the slide, I won't have time to tell you this, but one of our speculations that led us in this field has now been at least correlatively borne out by studies in England by Lenner Heaney in Holland and Gunel Biberfeld in Stockholm. That is, monkeys who are vaccinated who do get protected do produce high levels of chemokines. But let's go back to this hemophiliac story. So I already said they were exposed, actually intravenously inoculated with proven HIV-positive factor A. Some of these patients remain uninfected in spite multiple infusions. I'll tell you now, none of them had the double mutation for the receptor. But yet, on the contrary, the very first infusion with hepatitis C virus, they become infected. Something was weird about HIV, suggesting some responsible factor. Now this slide looks at over time this patient population. There's 128 hemophiliacs in the group. Notice that 14 to this day, it says, I have actually, it doesn't say to this day, but it's out to 1998, 14 remain uninfected to now. But more exciting to me and more impressive, we couldn't draw the number of infusions these people had for limitation of space. Although I wonder why the artist didn't make the arrow smaller. In any case, I mean, it just occurred to me. You may be wondering, why did that? Anyway, you can see that there are a lot of infusions. Notice after one year, only three people are infected. After two years, a rising number, but hardly all, look at several years after 84 only are infected out of 128, extraordinary. I repeat, they didn't have the receptor mutation. What they did have was the corollary. They're producing higher levels of chemokines than other populations we studied, not dramatically so. They're spontaneously producing about three times the amount as other populations. And we did a wide variety of possible controls. And if you say, well, how important is that if you study it in a lab? Actually, the people that are producing this amount inhibit HIV, but under the usual multiplicities of infection that we talk about in a lab, they partially inhibit, whereas under even high titers, this amount potently inhibits infection as completely as we can detect no detection of virus. So it leads to maybe some, oh, I forgot this last point. When are chemokines made? When you're exposed to something, it takes weeks, maybe a month, maybe even longer to make an antibody. To make a killer T cell, to fight virus-infected cells takes six to 10 days. To make chemokines takes only hours. We think they're one of the first lines of defense against HIV, quite by accident. And so we can summarize what we've said, that HIV here, shown in blue, with its envelope in dark blue, interacts with sugar molecules, not shown in the slide, because when the slide was made, we didn't know that, as well as the CD4 molecule shown in pink, and that that somehow brings it to the chemokine receptors shown in black, up and down. And then we know that a portion of GP120, called the V3 loop region of the envelope, GP120, is necessary to interact with that, and the virus will enter the cell via this pathway. But the chemokines shown in purple can block that. And we know that there are two kinds of receptors that are important. There are more that the virus can use, but for practical purposes, CCR5, CXCR4, and depending on the receptor, the chemokine, ranties, nipuan-alpha, nipuan-beta will inhibit or not. Our hypothesis is that resistance to HIV infecting is general, that is, to some degree, we all are resistant. And in a relative phenomena, maybe, maybe, firstly determined by the amount of HIV relative to the amount of the local production of certain molecules we call beta chemokines. Now let's go to the other side of this. The person is now infected. How do they progress more slowly to AIDS? And the first will show you people that have only one gene, one allele, rather, of CCR5 absent. From Steve O'Brien's work, the people who still get infected but they progress slower to AIDS with one allele for CCR5 gone. They express less receptors on their surface. It's a quantitative phenomenon, apparently. Well, how about if you produce more chemokines, do you progress more slowly to AIDS once you're infected? This is one of many examples of studies we are now doing. This one is again in collaboration with my friends Aguri. We have some that are going on in the instituting collaboration with people at Hopkins done blind, so-called max cohort, where people progress at different rates, you get samples blind and the correlation is striking. Done carefully by looking at cells, not serum or plasma, which you can't do properly. There's a clear cut correlation between people who progress and not, people who progress more slowly, produce more particularly of this chemokine, myth one beta. So we come to the conclusion that the expression of HIV suppressive chemokines and co-receptors correlates with protection from infection, a more favorable course of disease progression and the clinical data strongly indicate that the levels of the chemokines have prognostic value and that the molecules in theory can be used therapeutically to prevent or to treat HIV infection and that brings me to the last slide on these chemokines. Can we use them therapeutically? Now the first negative response to this would be, gee, they'll be inflammatory, they often promote inflammation and that might be too toxic. Second, maybe it's difficult to keep proteins in enough concentration, that's a logistic problem. And the third, maybe say that if you use these mip ones and ranties, wouldn't you drive it towards CXCR4 using viruses, the more pathologic? The answer to the latter is no, if you have one of the leels for the receptor and less number, you do better, you can already say that. However, these problems are in theory solvable. We have recently discovered that if we coat the chemokines with those sugars that you saw on the cell surface, if we take externally a chemokine, complex it with a component of the sugar, that it's half-life is better. It doesn't degrade as fast. That the antiviral activity goes way up and that the signaling or what would produce its toxic effects is lost. Therefore, we want to try to move this to the next step of development for possible consideration clinically. Now I'd like to move to a second biological approach and this comes out of studies of pathogenesis, how the virus causes disease. And it's very deeply collaborative. In fact, it's more led by, my friend Daniels agree, than myself. And the story goes back more than a decade and it looks at what does HIV do when it infects the cell? Now many people in the field have focused, including myself early on, too much on the HIV targeting the CD4 positive T cell and killing it and then maybe in time you become exhausted and the immune system goes down and that's AIDS. The phenomenon is far more general than that and more complicated. Indeed, HIV one infection of CD4 positive immune cells can lead to the death of that population of cells that are infected under certain metabolic conditions of the host. However, it's only a very small number of such T cells that get infected yet the bulk of the T cells cannot proliferate properly and not only CD4 cells but even CD8 T cells which are not targets of the virus undergo premature death and are relatively allergic. Problem is bigger so that after infection something happens to uninfected cells that diminishes their ability to form mature killer T cells, CTL, cytotoxic T cells and to produce beta chemokines for example so that they can't take care of well the infected cell population. It's this concept I think that receives too little attention. Now, we wanted to see years back if we could stimulate the immune system. Something like a company called Immune Response does with some dead pieces of the virus. We got frustrated that you could go so far and we came to the conclusion it was because some of these cells are relatively paralyzed. This was shown also by other workers and we wondered why and our speculations with Zaguri led us to two things. I'm not saying they're the only things important but we think these two are. One is a normal protein. It's called interferon alpha which I'm sure you all heard about. One would say interferon alpha, that's good for you because it blocks viruses, doesn't it? Yes, at some concentrations but at higher concentrations it's immune suppressive and inhibits proliferation that's why it's used in cancer therapy. It inhibits T cell proliferation quite nicely. We also had hints, HIV infected people are producing massive amounts of interferon alpha over producing it. There are other hints. People, excuse me, animals that don't get aged do not. Animals that do get aged do overproduce alpha interferon. We also had a second candidate in our minds. Now it's not a cellular protein, it's a viral protein. We talked about it before. It's called the TAT protein and I told you it was important in an infected cell for regulation of viral gene expression but some years ago, Barbara Ensley when she was a postdoctoral with me now in Rome and I found that this protein, oh excuse me, you got ahead of myself, can be excreted from cells and other groups, particularly Frankel whose work actually preceded ours, showed that TAT can be taken up by cells and still other groups including ultimately Frankel's group, our work and especially Arthur Pardee and his colleagues in Boston, showed that TAT taken up by an uninfected cell will induce energy and ultimate apoptosis of that cell. Okay now, going back to a previous slide, this shows T cell growth, normal, 100%. Let's say your cell's in mine but after HIV infection you see a big drop, 60 to 90% usually. Yet only a few percent of the cells are HIV infected yet the both are not normal. Other workers have shown as I already said that CD8 T cells which virtually almost never get infected also undergo a lot of death so they also are in trouble. We speculated that interferon alpha and TAT may be partially responsible in an important way for this and so we did these experiments in the laboratory that I'll now show you quickly. Here is T cell proliferation, normal 100% in white, HIV infected, you see a drop this time in 90%. What if we don't infect, we just use interferon alpha or TAT, you see that they are inhibitory of T cell proliferation. Actually they can induce, programmed cell death apoptosis of those cells. If we treat with antibodies to interferon alpha and TAT we can restore proliferation almost to normal. Based on these results and recent findings that in Europe in a blind study the best correlation in people doing well not looking at chemokines because that would require cells but using serum was the tighter of antibody to the HIV TAT protein. Based on that clinical trials were initiated in Europe and in Israel which we're bringing to Baltimore in the new year and this is actually vaccinating against a normal protein interferon alpha made in a modified form so it's not biologically active yet still immunogenic and against TAT. Think of TAT now as a toxoid like diphtheria toxin that you get vaccinated against the toxin sometimes. It's a toxoid, it works not on the infected cells only but at a distance. There's been four years experience now with vaccines against alpha interferon. It's safe, it's immunogenic and it's correlated with a stabilization of T cells even before therapy was available. Does not have an antiviral effect. The TAT work has only been started a year ago and I'm not in a position to be able to discuss those results yet they involve a lot of people other than myself but they are extremely interesting. Now why do I think about this for a third world? It's without toxicity. It doesn't require 30, 40, 50 pills a day at $15,000 a year. It's a few inoculations a year. We have hope that this can be brought to the less fortunate people in countries. Okay, that's the very last thing I wanted to tell you about and in the last couple of minutes I'd like to tell you about a finding that is honestly the most exciting thing I've ever been involved in. It's rather new but on the other side is uncomfortable presenting to scientific colleagues because I must tell you from the start though we have made some substantial progress in the purification of the molecule we are about a step away from its identity and so that's uncomfortable talking about something one does not yet know what it is except to say it's a small protein. And the second uncomfortable part is that I don't know the mechanism of anything because I have feared studying mechanism of something that's still in an impure state. That is we still have some contaminating proteins with whatever is the active molecule. Now this is a story that came out of total chance and luck. The story is a small protein present in the urine in the earliest days of pregnancy and we got onto this when we were studying the tumor of AIDS called Kaposi sarcoma. We had isolated some cells that could produce tumors in immune deficient mice. My coworker Joe Bryant who's head of our animal model division likes to say he made an aware observation. You could look upon it as he made a funny mistake. He was trying to study the male factor in Kaposi sarcoma, men get it much more than women. And so he was putting baby newborns, males and females over here. And he inoculated them with the cells. He came back in one set of experiments and he found that some of the animals the tumor didn't take. In some it was smaller than it should be. When he analyzed those animals he found that those that were in, that there were some of them are pregnant and he realized that he missed the genitalia in some way. And so what he discovered at that moment was that early pregnancy in the mouse was protected against this tumor from growing. A little later you don't protect but it slowed a bit. But if you look at late stage pregnancy there was no protection at all. An example of now doing the study more carefully is shown in this slide. And this has been published a few years back. So early stage pregnancy there's no tumor. You can see if the animals were pregnant later the tumor was developed. Non-pregnant animal obviously there was the tumor. And we wondered if this was a factor that could be found in serum. So we tested serum of mice and humans and we found that in early pregnancy there was a something that inhibited growth of Kaposi sarcoma tumor cells in vitro in the lab. You can't test serum just to get in an animal. And in addition to that when we looked later this activity seemed to be lost so that there's no real difference from the control. So this is a later stage pregnancy. And we wondered what could this be and we returned to textbooks and medical school under chronology, et cetera. And we were reminded that in the earliest days of pregnancy this molecule of hormone is made. It's called HCG, human chorionic gonadotropin which exists in these two chains which are not covalently attached. And in fact one chain is made alone in the earliest days of pregnancy and then the other is made and then the two together which are biologically active and it's used in the therapy of some pregnancies and for reasons I don't understand and understand the testicles. So we thought it must be this. So we went and we got clinical grade commercially available HCG and we tested it and it worked. So we were excited. And our clinicians, collaborators of ours immediately went forward and used it in patients and patients that were dying that were non-responsive to chemotherapy that had late stage A's with no T cells and they got some, this shows what happened in a mouse. There's the tumor and this is the tumor regress. You can actually totally prevent the tumor and then this is the patient and it shows, I don't know if you can tell but this would be a pleural effusion in this section. These are lung nodules and you get a great regression of the pleural effusion and then nodules by using this particular HCG. Now but if you'll notice this slide doesn't call it HCG, it says HAF because we have a tentative new name because during this period that the clinicians were doing this work, we discovered that some batches were positive and some batches were negative and you could never predict what was positive and what was negative. And we learned that what is commercial clinical grade that is used in patients is not HCG. It's a lot of things. It's filled with protein impurities. In virology and molecular biology when a bottle says something, usually it is what it is. It says in 1990s anyway but here apparently the patents were made 30 years ago and the way they prepared it is the way they still give it and that's it. And so what is called HCG is a lot of different things. We tested homogeneously pure HCG, it had no activity. We tested its purified chains, alpha and beta, no activity. The activity then purified to a low molecular weight protein. And now we have very few contaminating bands remaining. It seems to be a stable molecule. It seems to be a molecule which sticks to a lot of things suggesting it might have some fatty substance with it and now comes the extraordinary part of the story and I close with this. This molecule, when used clinically, they told me that in the Kaposi sarcoma they could sometimes see a drop in virus and a better rise in blood cell counts than they would expect. So we did a number of studies. I'm gonna show you one because of time. We call this HCG associated factor, we put it into monkeys. Monkeys that are susceptible to SIV, the monkey AIDS virus, and get AIDS in like six weeks time with a particular strain of SIV, okay? And we measured their weight, their T cells and virus. And this slide and the last slide I have is to show you that this is the amount of virus, untreated the virus is going up, untreated the virus comes down. This is now, what the hell is it? Weight, I think. Weight, I don't know. Can you tell me? What is that? Weight? Thank you. Oh yes, thank you. All right, so weight, you see the weight of the animals that are untreated, the two going down. The treated animals, the weight goes up. Now we come importantly to the T cells. And this shows the untreated animals, their T cells are declining as they develop AIDS. The treated animals goes up. Curiously, look at the bottom right. This is a controlled animal who's not infected. And so we gave him the same material and his T cells rose. Now what happens is that in addition to killing Kaposi sarcoma cells, we've learned that it also kills every prostate cancer we've looked at. But at the same time, we find it's a powerful growth factor for all the lineages of our blood cells. So it's either working at the stem cell level or it works on the early progenitors of our T cells, our B cells, our red cells, myeloid cells which give rise to granulocytes, platelets. They all rise. And we've done one biologic experiment in closing that I mentioned about that, that it has, we've been able to radiate animals. None of this actually has yet been published. We want to wait until we identify it to their death. 800 rads in a rat, 600 rads is the lethal dose for 50%. All the rats live and their bone marrow is completely restored. So it has an antiviral effect, a pro-hematopoietic effect, an antitumor effect. It concentrations five-fold higher than we've using. It's been without toxicity. We don't know what the heck it is. We suspect that this small protein is some embryonic factor that maybe in early embryonic life is there for purpose of shaping, like molding the fingers out of a paw, by killing certain types of cells, newly forming, blood vessels are killed, and that may be what's going on while promoting the development of the blood-forming system. Obviously, we want to run with this as fast as we can to bring it to practical use. To summarize, the HIV epidemic has plateaued in some areas and for some people, but in others it's getting worse. Predicting the future is not possible. I have expressed of you that there are three major issues now for AIDS reserves. First, to understand in detail while some people resist, why some people resist getting infected, and others who do not get infected, but remain virtually disease-free, even if untreated. I have emphasized that we know now at least one major set of determining factors to account for these phenomenon, namely the genetics of the chemokines and the chemokine receptor system on the cell surface, which are the gateway for HIV to enter cells. Second, the development of an HIV-preventive vaccine remains a top priority, though candidates are available, sufficient progress has not been made to assure us that they will be successful. No one knows when real success will occur. Lastly, though acknowledging great advances in the therapy of HIV-infected people, I've argued that research in this area cannot be lessened in order to focus on the development of a preventive vaccine because the therapies are imperfect, costly, and HIV-resistant mutants will likely occur, in some cases are occurring. Moreover, for the bulk of the third world and even for some people in the United States, such therapies are logistically difficult or impossible. I believe more simple biological approaches to treatment are needed and are likely to come from continued and patient studies of the basic biology of HIV. In this regard, I have used three examples of our own research. Two, I hope you will agree with me, came out of reasonably rational research approaches, and the last one, which was pure dumb luck. Thank you. What's that bandagenic, if you try to make anybody know? Yeah, actually, yes, we have been able to do polyclinic. Here, we have a napkin. Just started, yeah, no monoclonal yet. It's probably, I didn't see that at all. When are you going to be staying still? Are you still wired? Should I still be wired? Did you stay wired after your talk? They brought it off, what else? Flex off, be the radio then. Which is why what you're still closed now, that's not going to happen. Oh, boo, I don't know what you're doing. It's probably at the other end of the day. One hour? Two hours? How many hours? One hour. Yeah. Who's going to take the first? The person that's coming to visit me this afternoon, his name is Peter. Oh, good. I don't know the place. He's the specialist. I don't know what I'm going to do. What's that? What's that? What's that? What's that? What's that? What's that? What's that? What's that? What's that? What's that? Peter can figure out how to take the lavalier mic off. For the rest of my life. You can just leave it on if you don't need it. I mean, before I go to bed I'd like to take it off. We'll get it for you before then. Thanks, Jerry. I brought along a virus killer. Jesus, they were tearing at me. Did you guys have that trouble? Yeah. My nose. My eyes. We're ready now to, Dr. Gallo giving him a few seconds here to fuel up and recover. It's ready now to respond to questions that the other speakers may have raised in his wonderful delivery. John? Say something about patient toleration of these CC chemokines. Dr. Holland asked what about patients being able to tolerate chemokines? If that did get into the clinic in the future. One group I know in England has already tried chemokines, at least one, for other reasons. And I honestly don't know the toxicity data. But I want to say again that the toxicity as far as we know so far is due to the chemokine binding to its receptor and signaling. That you can mutate the molecule so it doesn't signal anymore, yet it's still antiviral. And better, if you simply coat it in glycosaminoglycans, then for reasons I don't understand, it's much more antiviral and yet loses all signaling. Okay, so we think that we could avoid substantive toxicity that way. The bigger problem is going to be the getting the sufficient amounts, I think. And I hope the sugar coating allows the degradation to be slow enough that it's achievable. Bob, could you possibly use a genetic approach so that you could insert the CDNA expressing chemokine and have it expressed by a particular cell type? That's a good point. I couldn't, but you could. Look, to be serious, I think it's an interesting idea, especially if you could regulate that expression. I don't see why you wouldn't think about this possibility. Along those lines, Bob, we can do that genetically a couple different ways. One would be to make cytokine that would be secreted, but we also are able to knock out the receptor gene in the recipient cell. Do you have a preference for which of those would be better? Well, you know, since I work more in the production of the chemokines, that my preference would be for you to work with us. I can't answer that. I think you could argue it either way, I think, right? One is a little more permanent than the other. Yeah, I think that the knockout more closely parallels the mutation that we see in human disease. On the other hand, I think the secreted factor may have more of what we'd call a bystander effect, and might affect more cells in trans, and so I could see some reasons to do it both ways. And if we could increase it to the level that the...let us assume that I'm right, that that correlation is meaningful. We can't say correlation improves causation, but when you have a mechanism that is there, you're naturally prone to want to believe that the overproduced chemokines are what is relevant. But let's say you could make the level just like in those hemophiliacs, instead of 8, 15, 40 nanogram per millimeter production locally. Bob, with respect to the HCG-associated protein, have you had a chance yet to look at its effect on T cells directly, or antiviral effects in fibroblasts against other cells? The answer to both is yes. If you study bone marrow from monkey, rat, mouse, and human, and look at progenitor cell populations, you see an increase in progenitor in vitro in all, treated with the almost purified factor. And so in vitro, you can demonstrate these kind of direct effect. The antiviral effect is a yes answer, but is an encumbered one. In vivo, the results are more dramatic and striking than I believe can be accounted for by what we see in vitro, strangely enough. For instance, I didn't want to get into all the nuances of this, but there is a mouse that is made transgenic for HIV. Not completely because you don't want the veterinarians to get infected, but partial HIV genome expressing most of HIV proteins. And those animals are born looking normal, but in a few weeks they fail to develop and they die with all kinds of problems. If we give this substance to the mother, let the animal feed on the mother's breast, those animals are completely protected against HIV expression and disease, and as long as that molecule is there, they're protected. So this says that a very low concentration is working, yet in vitro, you know, it's nanograms per mil. And I think we wouldn't be anywhere near that in those mice. So I think there's something that I have no idea what is going on in vivo. I have a question also about early pregnancy versus late pregnancy of an HIV human, HIV positive woman, and HIV newborn, is there epidemiological data to look at an HIV positive woman that develops perhaps the same time she's pregnant? She's already HIV positive and becomes pregnant? Is there some protection there? Yeah, it's obviously a very good question and one that comes up all the time. You know, about a third of women, the baby gets infected and I don't think anybody knows why the other two thirds don't get infected. So we've never tried to make a definitive study of that and I can't think of anything, I don't know any epidemiology that would give me an answer to your question, except there are two thirds of babies that don't get infected. Were their mothers producing more of this at the right time or was it just chance? I don't know. If anybody else knows more about that, who follows babies born with HIV or not, I could be helped. Question from the audience. Someone wants to know what role that nutrition plays in the development or susceptibility to HIV? Is there any contributing factor to nutrition? I don't know. And another question from the audience. Maybe somebody else knows, that's Gary. Question from the audience. Minnesota is looking to accepting more abstinence only teaching in the schools. That is no discussion of prevention. Is not education the mainstay of HIV prevention? Education as to how disease is prevented. You keep looking at me, I didn't talk about this, you know. All these guys could give their opinion on that, not just me. I think both, it's okay if you abstain, you know. I think what matters is important. For all aspects of knowing what the virus is, but there's not just HIV, there are other problems too. What matters is practice. Not so much education as practice. Now if education affects practice, then you can go either way. But if you're not going to abstain, you should know what else to do, I think. I think it's pretty clear that primary prevention is the mainstay therapy for many diseases. Cardiovascular diseases, it's becoming the case, but I think for HIV that may particularly be the case. And I think, CJ, what you've brought out is there's a difference between education and practice. But I think clearly, I think we observed in the gay community that as the fundamental facts of HIV transmission became apparent, there were changes in practice styles in that community with some tapering of the disease. So I agree completely that education is a critical component of curbing the infection. The reality is though that becoming monogamous or more monogamous has been part of that story. And almost all stories that have shown significant drop. It's not just, let's say, the use of condoms. A cell biology question about HIV, is it known which chromosome HIV inserts itself? Yep. Nothing special. They can insert a lot of different places. There's no magic chromosome for it. Right. Almost any. Yeah, it's just about anywhere. There are some hotspots and some people believe it. A question about the alpha interferon. There are a number of different alpha interferons. There's one in particular that's important, or is there something common among alpha interferons that is... Actually, the one that's... Alpha interferon overproduced by HIV infected people is supposed to be a somewhat... It's an acid-label form, somewhat structurally different. And I don't know if it's ever been chemically characterized as to how it differs. I don't see anything like that published. But the... No particular form of alpha interferon is anti-proliferative. I mean, they all are anti-proliferative as far as I know, at high concentrations. The concentrations, like, are produced in HIV-infected people. They can inhibit T-cell proliferation. So these are the levels that cause the aches and the pains and the malaise that you see and say influenza? I don't know. I don't know for some of the other clinicians. Or the release of TNF. TNF, tumor deprosis factor alpha. It's not just alpha interferon that's overproduced in HIV-infected people, but a number of what are called inflammatory cytokines. Another question from the audience, how stable is the HIV virus? In terms of this, I assume the binding, the multiple binding sites, what are the chances of it mutating again and thus negating the progress you've made? All the variants of HIV that we know of use chemokine receptors to enter the cell. Now, that doesn't say they're in variants that preferentially use another chemokine receptor. That's... And I cannot say that every variant on Earth uses chemokine receptors. Right now, everything that's been studied uses chemokine receptors. Now, I've given you two. Those are the main receptors used. It is true that there are other receptors, chemokine receptors, that some variants of HIV can use at a low level. Now, would you drive it towards those variants that can use other receptors? Ah, maybe. But we'll have chemokines for those two. Last evening, during one of the firing lines that was rather crowded, the one over in Olin Hall, a high school teacher asked a question about glycoproteins and the role sugars play in viruses. So I think you answered part of his question about viruses using not just proteins for gaining entry into cells, but sugars as part of that interaction between a host cell. Are there other viruses for which there is a... that use a similar kind of a strategy? Okay. So perhaps we'll buy some examples. Mixer viruses, paramixer viruses, rheomarsis on certain types of cells. Yes. The sugar portions on glycoproteins, in a number of cases, act as essential parts of this receptor. But to have this extra kind of bystander help. The sugars, I think Bob is really only scratching the surface of how important they are. Obviously, they're important in the receptor interaction, but they're really important in so many other ways. I think, for example, the viral envelope. When you have free virus circulating in the blood, we now know from the crystal structure of the envelope that essentially almost all of the protein determinants are masked by sugars. And so even though a person may make antibodies to HIV, essentially they never have a chance to access the virus and to neutralize the virus. So the sugars essentially form a shield from antibodies. I think at another level it's quite likely as well that with sugars, when they're put onto the protein as the protein traffics from the cell, actually help that protein find its way out of the cell so that it can't be presented to the immune system. So it actually provides a mechanism to escape from immune recognition. So I think Bob alluded to it very briefly in his talk, but I think there's a lot to look at there in terms of the problem. Not to be confused. What Gary's now talking about is sugar associated with the virus that is involved with the envelope. I didn't even mention it. Very different than the sugar on the cell surface so that both are critically involved, but for very different reasons. Oh, okay. Apparently there are a large number of males over 50 at Nobel that are interested about this HAF therapy and prostate cancer. Thanks. I thought the question was going to go elsewhere for another drug. That was surely going to save it. I was going to ask him to ask you. I didn't mean that. I'll take that back. Okay. We haven't fully explored every type of cancer. The only reason I commented on prostate is because we did do, I think, enough to say it works in a laboratory setting, meaning that each of the transformed prostate cancer lines we looked at underwent programmed cell death when treated with this. In addition, we had access to two or three primary biopsies, and the same thing happened. In addition, we have a few animal models, some in our institute and some in collaboration with Dr. Papas in Charleston that has worked in vivo in the transplanted tumor cells to the mouse. We've looked at some cancers where there's been no activity, like some lymphomas, some leukemias, and very, very quickly looked at a few from breast cancer where there was some activity. As soon as the molecule is pure and identified, the two things we want to do is explore mechanism, mass produce it, and really intensify the study in cancers and get it into therapeutic trials in AIDS, in the cancers, and obviously maybe to increase the dose of radiation to a person who's getting radiation therapy but has toxicity problems. All these things need to be properly evaluated. For the latter, we have a collaboration with the U.S. Navy in their institute directed towards things like this, and we'll be supplying material to them. I extremely correlate with the age group you're talking about and then quite a bit of some, so I'm interested too. We'd like to get it going forward as quickly as possible. It requires finishing the purification, its identification, a little bit of mechanism of action, large-scale production, FDA approval, et cetera. I think also, Dr. Gallo, that the implications for this type of factor really are quite broad for medical diseases and may extend beyond the cancers. So if this is some type of a stem cell factor that promotes hemopoietic or blood stem cell proliferation, it might be very useful for some of the anemias or so some of the chronic autoimmune diseases potentially as well. So there may be much broader implications. We'll have a great difficulty trying to name this. There's peptide in there. Dr. Yacou has a question or comment. Bob, what your discussion today has brought up is the extraordinary complexity of cytokines. I mean cytokines are the intercellular messengers that cells tell each other what to do. And we're at the very beginning of knowing what they do. They all have very multiple effects. Like, for example, as you quite rightly pointed out, some of the ill-feeling influenza is due to interferon, yes. TNF is a name that we give, but there's many more activities than just killing tumor cells. And so this particular factor, this particular probably cytokine that you've discovered may have specific activity but probably has many other activities also. And we're just at the very beginning of understanding of it. A comment back. I certainly would agree with that very much. Once I was pressed in Singapore, to people in Singapore to try to predict what medicine was going to be in the next century. I can't begin to do that. You know, you retreat into the human genome project or something. But I said the one thing I felt confident about or at least what would be needed would be divisions or departments or subdivisions of medicine of cytokinology. Because, yeah. I have a question of you as a retrovirologist. We all carry footprints of retroviruses in our chromosomes. They are defective. They aren't being used. Is there speculation on origin or perhaps they are playing a role in human development or not? Yeah. I think this could be punted around the table in most directions also. But the way I look at it is that, as you heard from John yesterday, that some of these things evolved with us and much before us. And some of the ones that animals including us have within us seem very likely to have been infections that hit our germ cells and seem to have been maintained. When things are maintained, you generally think it's for an advantage. So I kind of see this, and I once wrote about it in a conceptual way in the book that I wrote, is that it's like an endless thing as the eons go on among the species that carry these viruses and the virus sequences themselves is that if you infect the germ line, if it's useful, eventually it becomes part of our genes that are maintained with us, sometimes it evolves into a whole viral particle. Sometimes it's only a piece of the viral genes that survive. And when it evolves to a whole viral particle, sometimes it evolves further that it can infect. And sometimes it infects some other species. And so an endogenous viral sequence becomes a virus particle, became a virus and starts to chain over and over again. And it's in the process of when it became an exogenous virus infecting a new species early before adaptation that you generally see disease. What does it really do for us? Does it have anything to do with speciation or cell development, differentiation? Howard Temen used to speculate, delayed Howard Temen a lot on such things and I tended to be one of the people following that Pied Piper for a while and I used to like to read speculations on it. But they were in this kind of direction but now there's even evidence that things that Gary referred to as those last night, others have referred to so-called retro-transposons that are active in all of us and they're at the beginning of becoming a retrovirus in a way. They're part of a retrovirus and they actually shift sequences around in our chromosomes that have something to do with regulation of other genes probably. But why do they evolve to escape? Is that an accident? An advantage when they jump species? Is that sometimes an advantage in varying species in some way? The last time is very fantasy. Well this relates to what perhaps the human genome project will tell us as we've known for quite some time. The vast majority of human DNA does not encode proteins. I mean it has no function. It's the part of DNA that Francis Crick many years ago called junk. Does it have a function or not? There is some interesting recent literature on this idea of transposable elements and human evolution. It's actually rather striking to me that despite the numbers of cases of HIV that we've seen around the world that we have yet to see HIV integrate into our genome and be passed on in the germline. It's really related actually to the factor that you're seeing early on in pregnancy that maybe there's a protection against that. In contrast, the retro transposons which are really a different class of retroviruses, their reverse transcriptase is more like our telomerase that primes off of the DNA of our chromosomes. There actually has been a recent report from Hayek Azantian at the University of Pennsylvania that arose from the insertion of a transposable element into one of the hemophilia genes and it's a new mutation and there's active evidence that he can point to the generation where this new mutation arose and where it came from. It came from this line element that's such retro transposons. So clearly those elements are involved in human evolution as we speak and there's hard evidence for that. So retroviruses, they're in our genomes as Bob said. Probably at some point they are doing something. It is curious though that we haven't seen HIV. Well, you all have been sitting there very patiently for an hour and 45 minutes and the Dr. Gallo has been on the spotlight for an hour and 45 minutes so it's time for lunch break. We will readjourn at 1.30.