Page History

Ronald Desrosiers: [00:00:00] Okay, thank you. This sounds like it's working. It's an honor and a pleasure to be here. First, let me say could you add those seven minutes to my allotted time? 

I'm going to be talking about the discovery of SIV and the development of monkey models for the study of HIV/AIDS. Let me just say upfront, apologize upfront to [00:00:30] anyone whose name I inadvertently omit, or for contributions that are not part of my presentation.

The story I'm going to tell tonight is largely a personal one but I will try to interject as many anecdotal stories as I can. The story for me begins with this landmark New England Journal of Medicine paper that's been discussed [00:01:00] a few times so far at this meeting from [Michael] Gottlieb et al. (1)

I remember like it was yesterday, the director of the New England Primate Center at the time, Ron Hunt, walking around the halls of the primate center waving copies of this article saying, "This looks like what our monkeys have, this looks like when our monkeys have," and indeed it did. There were monkeys dying at the New England Primate [00:01:30] Center with wasting syndrome, opportunistic infections, pneumocystis Carinii pneumonia, generalized CMV. At some point over the course of the next year or so, I decided to devote a percent of my effort. I was working exclusively at the time on herpes viruses. I decided at some point over the course of the next year or a year and a half, to devote a percent of my effort to see if I could define [00:02:00] viruses that might be the cause of this immunodeficiency. 

The original description of that immunodeficiency syndrome from the New England Primate Center appeared in Science in 1983, Norman Letvin (1949–2012) et al describe this disease, again remarkably similar to the GRID disease in the New England Journal paper. (2)

Around the same time, there was a [00:02:30] similar description of similar disease occurring in rhesus macaque monkeys at the California Primate Center, including Roy Henrickson, Murray Gardener, and others. (3) Over this period of time, the Davis group and the New England group were real competitors, so we were competing with each other constantly, but it was a very friendly, wholesome competition, and [00:03:00] one that we still love to talk about to this day. 

My group spent some time following a number of leads. Particularly we got a number of fresh CMV isolates from these monkeys with immunodeficiency, did some characterization of them but it just didn't make any sense that it was that. Then in 1984, [00:03:30] I think just a few months before the Bob Gallo and coworkers four publications in Science magazine (on May 4, 1984, see 4, 5, 6, 7), we published a report of the isolation of new type D virus associated with this immunodeficiency syndrome. (8) And indeed, this type D virus was the major cause of morbidity and mortality in our macaque monkeys. Also around the same time, our competitors [00:04:00] at Davis, California published a similar report in Lancet. (9) I, of course, derived great joy in pointing out wherever I could, how we published it first, February 10 versus February 11th. I actually did like a news and views cover story as in the New England Primate Center newsletters, pointing this out. Preston Marx in all in good fun took objection and pointed out that well, [00:04:30] where the Lancet is published is six-hour time difference with East coast. It's a nine-hour time difference with California so they were actually published at the same time. I think this is an example of the good fun that we had going back between Davis and New England around that time.

Now, Davis continued to aggressively pursue the type D virus story [00:05:00] and its causative role in the immunodeficiency. We decided to take—we took a largely different course, and credit for taking that different course goes largely to Norval King. Norval King was the head pathologist at the New England Primate Center but he was also an expert electron microscopist, who really understood the differences in morphogenesis and morphology of the [00:05:30] different subgroups of retroviruses. He was doing extensive electron microscopic examination of sections from lymph nodes of these monkeys.

He had pictures which he recognized and told us was of the lentivirus subfamily, basically budding from the cell membrane without a preform nucleoid and material particles [00:06:00] with a cylindrical or rod-shaped nucleoid. We went hunting for trying to recover this virus. We were encouraged along the way. Now I'm not sure this was the first morphologic relatedness of the human AIDS virus but we were encouraged by this publication in 1984, with [Françoise] Barré-Sinoussi, [Jean-Claude] Chermann, [Luc] Montagnier, and others, relating the [00:06:30] morphogenesis and morphology of their LAV virus with the equine infectious anemia virus (EIAV), the horse member of the lentivirus subfamily. (10)

Lo and behold, we were successful in recovering this virus with the lentivirus morphogenesis, which we published in June 1985, in Science with Muthiah Daniel (1927–2018) as the first author and myself [00:07:00] as the senior author. (11) I think this was a little more than one year after the four publications in Science from the Gallo group on what was called HTLV-III in their publications. This lentivirus that we described in this paper was not the major cause—it was a cause, but it was a minor cause of the [00:07:30] immunodeficiency in our colony. Nonetheless, still a cause.

We hooked up with French researchers, principally Marc Alizon and Pierre Sonigo for the sequencing and their sequencing revealed it to be closely related to what is now called the HIV-2 virus. (12)

Subsequent to [00:08:00] that time, subsequent to our 1985 publication then, over the subsequent years, there was a whole spate of reports of isolation of SIVs from a variety of African non-human primates, including African green monkeys, and sooty mangabey monkeys. I show this paper from Vanessa Hirsch and Phil Johnson because I think it was the first or one of the first [00:08:30] to show that the virus from sooty mangabey monkeys, SIVsm was basically a member of the same family as our SIVmac virus, and a member of the same grouping family as HIV-2. (13) So, HIV-2, SIV sooty mangabey, and SIVmac for one grouping are closely related viruses. [00:09:00]

I'm not going to go into this in any detail. I think subsequent speakers in this session are going to cover extensively the wide range of African species that are naturally infected with their own SIV. I find a couple of points interesting though that I would like to make. The first is that Asian Old World primates do not appear to naturally harbor SIV. It's something that's peculiarly found [00:09:30] in African Old World primates.

The second point that I would like to make that I'm a little surprised hasn't been studied over the years and that is that baboons do not appear to naturally harbor their own SIV. Considering the wide range of African species, I don't know if there are any other African species that have been found not to be naturally infected. [00:10:00] To me, this is a real curiosity. Is it the collection of restriction factors that they have? Are they really strong restriction factors? I don't know. In this publication, we surveyed sera from 279 baboons that were gathered by Jeff Rogers, in the lab he was in at Yale at the time, from Tanzania and Ethiopia. (14) They were almost all negative except two [00:10:30] baboons that had serologic reactivity that was strongest with SIVagm. We pointed out in this paper that baboons are known to occasionally prey upon and eat green monkeys in these natural habitats in Ethiopia and Tanzania.

That's where [00:11:00] Beatrice Hahn got involved. I think she called me and we provided sera to her. Is that right Beatrice? Yes. Beatrice used her outstanding sequencing capabilities, and indeed showed that the sequences present in these two baboons were of the SIVagm of the vervet subtype, so indeed they were infected with SIVagm. (15) It does not appear to be a natural infection of these baboons and it made a very interesting curiosity [00:11:30] why that would be. 

Keith Mansfield, when he was at the New England Primate Center, worked pretty hard to try to figure out, where did this SIV come from? (16) It had become apparent that macaques are not naturally infected in their natural Asian habitat. Sooty mangabeys are. Where did this SIV come from? [00:12:00] He was able to trace back the origins of SIV at the New England Primate Center to a shipment of six monkeys that we received from the Davis, California—yet again, something else we can pin on the Davis group—that we received from Davis in 1970—did I say six? it looks like five—all of whom turned out to be infected with SIV. [00:12:30] Infected a monkey that was born in New England, monkey MM, Macaca mulatta rhesus monkey 7872, who developed a lymphoma. And Ron Hunt and Norval King said, "Okay, let's see if we can pass that lymphoma,” and passed it to a monkey, 251, which is the origin of SIVmac 251 that many laboratories use.

That animal, [00:13:00] I believe also developed a lymphoma, but then the subsequent passages were not lymphoma, but was clearly AIDS disease, and including another one, 239, which is the origin of the SIV 239 virus.

So, where did the SIV come into the California macaque monkeys? That has not been definitively shown but rumor has it that Carleton Gajdusek (1923–2008) [00:13:30] at the time was doing transmission experiments, trying to transmit prion diseases and going one species to another, with inoculations into brain and inoculations in general. That very well could be the origin of transmission from sooty mangabey monkeys, to the rhesus monkeys in Davis, California.

The next step on this personal [00:14:00] journey for me is the definition of an infectious molecular clone of SIV that could be used for laboratory purposes. This was published in 1990 in Science, after several years of the Scientific Advisory Committee advising me not to try to do this. (17) It was a hopeless task but we ended up being successful at it. [00:14:30] This (SIVmac239) turned out to be not just the first infectious pathogenic clone in the SIV/HIV group of viruses, but actually, the first infectious pathogenic clone to be defined for any lentivirus. Having a clone virus and now a number of different clone viruses that are fully pathogenic is enormously useful for a variety of applications, [00:15:00] most of which were listed by Jeff [Lifson].

At the time, however, of this publication, the viral etiology of AIDS was being questioned—Peter Duesberg et al., as we heard mentioned earlier in the meeting. Our report of the induction of AIDS by cloned DNA of defined sequence was used as unambiguous evidence for the viral etiology of AIDS in monkeys, and thus [00:15:30] consequently in humans as well. Use of this clone is enormously useful for a variety of purposes, is a defined sequence, 10,279 base pairs in length, one can change any nucleotide, any series of nucleotides, any gene, take it out, put in a different sequence and ask the consequences of those changes.

It provides a [00:16:00] homogeneous virus of defined sequence for studies, for a better-controlled laboratory setting. This is especially important in vaccine studies, where oftentimes, the heterogeneous mixture present in an uncloned virus stock can create complications for interpretation of the results. As I said, any gene, any nucleotide, any amino acid can be changed at will, and the effects on pathogenic potential, tropism, neutralization sensitivity [00:16:30] et cetera, can be determined.

Currently, there are now a variety of different SIV and SHIV models that are being used, and I'm not going to go into those in detail, but I've listed here what I think are the principal uses of these SIV and SHIV models. First is the better understanding of pathogenesis. If you have a question about the role [00:17:00] of macrophages and persistence, macrophages and reservoirs, the contributions of auxiliary genes, etc. etc., one can define experiments using SIV or SHIV models to address the question in ways that would never be possible in humans. These models are also important to inform and guide the development of vaccine concepts [00:17:30] for development, for use in humans. It's really not just that it's vaccines but it's also novel approaches to therapy and novel approaches toward cure strategy, and that's listed as the third point here.

I may not even need those seven minutes back. In the time that remains, I would like to cover [00:18:00] a current use of these model systems in my laboratory, and it's a use that I'm particularly excited about for its potential for having a real impact on the HIV problem in people. That is the use of adeno-associated virus, AAV, as a vector for delivery of monoclonal antibodies for the prevention of HIV infection and/or [00:18:30] the treatment of HIV infection. I personally, and many in the field feel that there is a need for alternate strategies in the vaccine prevention arena. There's a need for a variety of alternate strategies toward a real cure, a hot topic these days. And I think long term delivery [00:19:00] of monoclonal antibodies is one such promising alternate strategy. Such antibody-based strategies are made possible by an incredible array of monoclonal antibodies with potent, broadly neutralizing activity, that have been isolated and characterized over the last several years. A variety of labs have been involved in the isolation and characterization of these monoclonal antibodies, including Dennis Burton, [00:19:30] who's sitting here—wave to the crowd, Dennis—Michel Nussenzweig at Rockefeller University, who is not here, the VRC (NIH Vaccine Research Center) group, and others have done an amazing job in isolating a wide variety of such antibodies.

One can, of course, deliver such purified antibodies passively, but as we try to look forward to long term solutions [00:20:00] I think we need to need to be able to deliver these antibodies in the long-term, for very long term. I think AAV vector is ideal in many respects for achieving this. The AAV vector system, the only protein expressed from AAV comes from the transgene that you put into it. As long as that transgene product is not viewed as foreign, one can get very long-term expression, particularly from muscle cells, from intramuscular [00:20:30] inoculation of the desired antibody. There is such a proven ability for long-term expression of the transgene product even in human studies, in studies that have used AAV vector to correct—to replace proteins missing in hereditary disorders. There's an outstanding safety record in human gene therapy trials. In fact, AAV [00:21:00] has become the vector of choice for gene therapy experiments in humans. Because of the nature of the vector system that I won't go into, there is no integration of the AAV vector DNA into host genome sequences.

I'm going to show you two examples of results from my lab. The first is on the prevention side. This is one of the first monkeys that we did. [00:21:30] Single inoculation on day zero of AAV vector, AAV1 given intramuscularly delivering the rhesus monkey monoclonal antibody 5L7 and IgG1 form. We're now out more than three and a half years. Three and a half years and 250, 300 μg/ml steadily delivered three years. Just try to envision [00:22:00] human use around the world: one day, one inoculation of one AAV making one monoclonal antibody, and the other arm, inoculation of AAV making a second antibody. You basically have protective levels, sterilizing immunity for decades, or for the rest of your life, with the right antibodies consistently delivered. [00:22:30] That's the vision. I think it's possible.

On the therapy front, or toward a cure front, I will show you one more example from my lab. This is monkey 2438 infected with SHIV-AD8 peak viral loads around week two, viral load set points done in Jeff Lifson's group in Frederick and the ten thousand twenty thousand range for 86 weeks, never on antiviral drugs, [00:23:00]given AAV1, making two antibodies, Michel Nussenzweig's antibodies, 3BNC117 and 10-1074. This animal has now been with a single AAV inoculation, never on antiviral drugs, suppressed to below detection for more than a year.

Again, envision this for use in humans. [00:23:30] You could start with say someone who's effectively suppressed on antiviral drugs. They come in one day, one arm, AAV one—AAV making one monoclonal antibody, AAV making a second monoclonal antibody. You deliver therapeutic levels. They go off antiviral drugs and they continued to be suppressed. Again, in the absence of antiviral drugs. Again, I think this is not an unrealistic scenario for use in humans [00:24:00] and I am really excited to try to see this through to completion. I think with all the talk toward a cure, what can we do to actually totally rid HIV? I think this is a new tool. A new—something new in their armamentarium to try to achieve that. [00:24:30] 

Here you have the antibodies. These are IgG1 with good ADCC activity. Any cell in the reservoir that tries to start making viral proteins and making virus can be killed by the circulating levels of antibodies. Here I just showed the two antibodies that we delivered, blue is 10-1074. They're in, I think the 100, 150 μg/ml range. We're now out over a year and effectively delivering [00:25:00] these two antibodies.

What's the catch? Well, the catch is all the monkeys don't behave this way. (15) The problem that we have faced is the development of antibody responses to the delivered antibody that severely limit the concentration of the delivered antibodies that can be achieved. Most of the work in my lab in the last three years has been looking at a variety of approaches to try to [00:25:30] minimize that problem, minimize the anti-antis and consistently deliver the antibody of choice. I think we're pretty much there now.

This is the last slide. I think everyone in the room here can be proud of their contributions they've made over time, but I'm not satisfied to where I am. [00:26:00] I think probably most of you aren't either. We've got a ways to go. For me, it's seeing this AAV approach to completion where we can create a sterilizing barrier to infection with one inoculation for the rest of individual's life at a low cost. We can achieve a drug-free suppression in already infected individuals. I think those goals are achievable.

I'll stop there. Thank you. [00:26:30]

[applause]

Jeffrey Lifson: Thanks. I think we have time for a couple of quick questions that we have some, while we're waiting for questions and the microphone to be passed, I'll just say that as someone in keeping with some of the other remarks through the meeting, as someone who came to non-human primate AIDS model research relatively mid-career, I just wanted to compliment Ron for his many contributions to the field, his generosity with our agents and expertise. [00:27:00] As you can tell from his talk, he's continuing passion and enthusiasm to deal with this problem in the setting in which he's chosen to address it.

Wasif Khan (University of Miami): Yes, wonderful talk. I'm wondering, well, if it becomes really successful. Is this a better approach than try to make various constructs and designs for vaccines, which will certainly have some variability in human population and here you're providing something which is already working [00:27:30] or will be, once it's—How do you see, is this a better approach or how do you compare basically the two approaches?

Ron: I think many of the people in this audience know that I, over the years, have seen and spoke about the enormous barriers to making a vaccine against HIV in the classic sense where you deliver an immunogen, you're having an immune response and you hope that immune response is protective. [00:28:00] It's going to be enormously difficult. I think vaccine trials in monkeys and vaccine trials in humans have pretty much born that out, it's going to be very difficult.

I think this approach—my lab is still working on that using recombinant herpes viruses, I've not given up. But for me, it's much easier to envision the AAV delivery of antibodies having a potential of working. We already have these antibodies. They're potent, [00:28:30] they're broadly neutralizing, and if we can figure out a way to deliver them long-term, it can solve the problem. No one knows how to induce these kinds of antibodies with an immunogen. It's worth trying, Dennis [Burton] and others are figuring out ways to try to tease the immune system there, but this approach circumvents the problems.

Raymond Schinazi: Very exciting results that you have at the end. I wanted to know how much transduction you actually get with the AAV and whether using AAV-1 [00:29:00] or AAV-2, which I haven't read your paper. I'm afraid.

Ron: How much transduction. What percent of the DNA molecules actually get taken up? Boy, I have no idea of how one would measure that.

Raymond: Yes. I was just wondering if you can give a clue—if it's possible.

Ron: In terms of the AAV serotype, that's a good point. That is one of the things we've been looking at over the course of the last three years. If we change the serotype that's used once with different properties, can we alter the results? [00:29:30] AAV-8 has been a virus that has been reported to preferentially expressed in liver. Liver expression has been reported to be tolerogenic. With our most recent changes to vaccine design, including the incorporation of micro RNA target sequences that shut off expression and antigen-presenting cells like dendritic cells [00:30:00] and use of AAV-8, it looks like at least with the 406 antibody that we are consistently avoiding the anti-anti responses and consistently delivering therapeutic levels of the antibody.

Raymond: So you could take a liver biopsy from the animals and see how much it's being transducted.

Jeffrey: Quick question from Bart [Haynes] and then John and move along. [00:30:30]

Barton Haynes: Could you bring us up to date on (a), is there a need to be able to turn on or turn off the AAV and (b), if there is, how you'd go about that?

Ronald: I think at this stage, we're trying to keep it simple, we're trying to get it to work in monkeys, and work with people who know how to do human trials. We've seen no safety problem. The [00:31:00] AAV work that's been done so far in humans, it's been beautifully safe, and as I said, it's a vector of choice. If safety problems do develop, one solution is on-off switches. Michael Farzan at Scripps in Florida is working toward that direction. We've not worked on that, we're doing simple straight delivery. What was the second question?

Bart: Well, that was it. Is it needed and if it's needed, what's [00:31:30] the mechanism?

Ronald: I would say now it's not, whether the FDA would feel that way, then [crosstalk] the FDA say you're going to deliver for 10 years, you're going to need a safety switch? I don't know.

Jeffrey: John.

John Coffin: Two practical issues. One is, what is the dose of AAV that you now need, and is that practical in terms of expense? How far off is that from what you would need to be able to do this practically in humans?

Ronald: It's comparable to the doses that are used in human [00:32:00] gene therapy trials. I forget off the top my head, it's 10 to the 12 vector genomes per monkey, something like that. Is it practical? Yes.

John: In practical terms, almost certainly given the likely much greater diversity actually in the HIV reservoirs compared to what it is in your monkey models, how many different antibodies do you think you would need to have to do this and to a working—?

Ronald: That's a good and important question. [00:32:30] It's actually very surprising to me that we got complete suppression for over a year with just two antibodies. I felt, most people in the field would feel, you need more than two, there's is going to be escape. Based on a publication from Michel Nussenzweig a year or so ago, there is evidence that escape from this particular combination of two antibodies can happen. [00:33:00] But the virus that's the escape virus is a tier 1 virus that's very sensitive to neutralization.

So in the context of therapy, in this case, 86 weeks of infection, where the animals have been making antibodies for 86 weeks, I think the explanation for our result, with no escape and complete suppression for over a year is that the escape virus is so deficient, [00:33:30] so sensitive to neutralization that it doesn't grow on.

Jeffrey: Thank you very much, Ron.

[applause]

[00:33:42] [END OF AUDIO]

Citations

Index

• 1.5 John Coffin — The Origin of Molecular Retrovirology 
• 2.0 Michael Gottlieb — Introduction to Session 2 
• 2.4 Robert "Bob" Gallo — Discoveries of Human Retrovirus, Their Linkage to Disease as Causative Agents & Preparation for the Future 
• 2.5 Françoise Barré-Sinoussi — Discovery of HIV 
• 3.4 Raymond Schinazi — Discovery and Development of Novel NRTIs 
• 4.0.1 Jeffrey Lifson — Session 4, Introduction 1 
• 4.3 Beatrice Hahn — Apes to Humans: The Origin of HIV 
• 6.2 Dennis Burton — How Does HIV Evade the Antibody Response? 
• 6.4 Barton Haynes — Development of HIV Vaccine: Steps and Missteps 
• adeno-associated virus (AAV) 
• African green monkeys, vervet monkeys (Chlorocebus) 
• Alizon, Marc 
• baboons (Papio) 
• blood — banks, donors, plasma, screening, transfusions, clotting factors (factor VIII), PBMCs 
• California National Primate Research Center 
• Chermann, Jean-Claude (b. 1939) 
• credit, priority 
• cure vs. remission of HIV/AIDS
• cytomegalovirus (CMV) 
• Daniel, Muthiah D. (1927–2018) 
• dendritic cell
• Duesberg, Peter H. (b. 1936) 
• early names for AIDS — gay cancer, gay pneumonia, GRID, 4H, KSOI, slim disease, etc. 
• early theories of AIDS etiology 
• epistemic object becomes the technical object 
• equine infectious anemia virus (EIAV) 
• Ethiopia 
• FDA (US Food and Drug Administration) 
• Gajdusek, Daniel Carleton (1923–2008) 
• Gardener, Murray 
• Henrickson, Roy 
• herpesviruses 
• Hirsch, Vanessa M. 
• HIV vaccine 
• Hunt, Ronald D. 
• iconoclasm in science 
• Johnson, Philip R. 
• Khan, Wasif N. 
• King, Norval W. 
• Lancet (journal) 
• Letvin, Norman L. (1949–2012) 
• leukemia and lymphoma 
• lymphatic system (lymph, lymph nodes, etc.) 
• macaque, rhesus macaque 
• Mansfield, Keith 
• Marx, Preston A. 
• microscope — electron and optical 
• models (model systems, model organisms, modeling) 
• molecular cloning 
• monoclonal antibody 
• Montagnier, Luc (b. 1932) 
• New England Journal of Medicine (NEJM) 
• New England Regional Primate Center 
• NIH Vaccine Research Center (VRC)
• Nussenzweig, Michel C. 
• Old World monkeys (Cercopithecidae, Catarrhini) 
• pathology (medical discipline) 
• Pneumocystis pneumonia (PCP) 
• prevention of HIV/AIDS
• prions 
• restriction factor 
• retrovirus classification, subfamilies, and genera 
• Rockefeller University 
• Rogers, Jeffrey 
• Science (journal) 
• scientific competition and collaboration 
• Scripps Research Institute (TSRI) 
• sequencing 
• simian-human immunodeficiency virus (SHIV) 
• simultaneous discovery (multiple discovery) 
• Sonigo, Pierre 
• sooty mangabey (Cercocebus atys) 
• Tanzania 
• tropism
• UC Davis, UC Davis School of Medicine 
• Yale University, Yale School of Medicine