Milestone: Dr. Robert C. Gallo and the Discovery of HIV-1

An interview by Dr. Paolo Lusso

National Institutes of Health (NIH), Bethesda, MD, USA

In this issue of the journal, we inaugurate a new series entitled “Milestones”. This series will encompass interviews with some of the pioneers that have laid the foundations of HIV research. Revisiting these landmarks while taking into account the prospect of their founders should be an inspiration to our readers, particularly the youngest generation. Nobody better than Dr. Robert Gallo could be the protagonist of the inaugural “Milestone”, with his recount of the discovery of HIV-1, the causative agent of AIDS, and the development of the first blood test to diagnose HIV-1 infection.

Robert C. Gallo is the discoverer of the first human retrovirus, HTLV-1, and the co-discoverer of HIV-1. Among his many seminal contributions are also the discovery of IL-2 and the development of the HIV blood test, which has helped save millions of lives worldwide. He has received innumerable prizes and awards, including two Lasker Prizes. He is the co-founder and director of the Institute of Human Virology (IHV) in Baltimore and the co-founder and scientific director of the Global Virus Network (GVN). He was the most cited scientist in the world between 1980 to 1990.

Dr. Gallo: Thank you so much for accepting to talk with us about your discovery that started the entire field of HIV research, which is the namesake of our journal. Last year, we commemorated the 40th anniversary of the publication of a short report in the CDC MMWR bulletin, describing a few cases of atypical pneumonia in homosexual men in Los Angeles. What were your thoughts when you first heard about this new disease? Did you immediately think that the causative agent could be a retrovirus? Honestly, I did not pay much attention to it. It could have been just a cluster of uncommon infections like many others that remain totally inconsequential. And no, I did not immediately think the underlying cause could be a retrovirus until I started to really think about it.

But then, at some point, the new disease did catch your attention. How did it happen? One day in early 1982, Jim Curran from the CDC came to the NIH to give a lecture on the epidemiology of these unusual infections and presented a clear picture of the risk groups: they were the same as for HTLV-1. This is when I started to really think it could be a retrovirus. It was a sunny day. After his lecture, we walked back together toward Bldg. 37, and he told me: “The epidemiologists have done their job. But where are the virologists?” That was a turning point for me. In the following months, clinicians started to send us patient samples from different cities and we started to culture their T cells in search of an HTLV-1-related retrovirus. The first experiment in our lab books is dated May 1982.

Was the idea of a human retrovirus well accepted at that time? Now it seems to be common sense, but back then, the idea that AIDS could be caused by a retrovirus was very controversial. Not to me, of course, after the discovery of HTLV-I and -II. To me, it was pretty clear that this was not an acute respiratory virus or an acute virus at all. When a virus is dripping off your tongue, the cause of the disease is obvious. But this was a hidden virus, a mysterious agent of a mysterious disease, and there were all kinds of theories on what the cause could be, including certain behaviors or drugs. Plus, the patients had so many other microbes replicating in their bodies that it was really hard to pinpoint the one that could be the cause of the disease. I had long discussions with my good friend Max Essex. We were reasoning that it had to be something new and that it was likely transmitted through biologic fluids like blood, or sex and motherto-child transmission, which we had already seen previously with HTLV-I and HTLV-II. Also, there was a high prevalence in Haiti where HTLV-I was also prevalent. So, the retrovirus idea became predominant in my mind, and the tropism for CD4 T cells strengthened that idea.

And then what happened? Then, one day in late 1982, Jacques Leibowitch, a French clinician at Raymond-Poincaré hospital in Paris who later became my friend, walked into my office. He had listened to one of my lectures and landed in the US with a dry-ice box containing 5 frozen blood samples from people with AIDS. He thought that my idea of a retrovirus was right and that I should test those samples for HTLV-1. I told him that my prediction was that we would find a variant of HTLV-I with changes in the envelope region that could alter the biology of the virus, which of course was not exactly true. It is ironic that Leibowitch was the 1873-4251/23 © 2023 Bentham Science Publishers Interview Current HIV Research, 2023, Vol. 21, No. 1 3 person who also got the Pasteur group interested in studying AIDS. After returning to France, he met the Head of Pasteur Diagnostics, Paul Prunet, and told him about our projects. In turn, Prunet talked with the scientists at Pasteur and suggested them to follow our ideas. Now, one of the samples that Leibowitch brought to us was at the same time a fortune and a setback. It was the blood of a young Frenchman who was in Haiti for his honeymoon, got involved in a bad car accident, and received a blood transfusion. He later developed AIDS, but the blood he received was contaminated by both HIV-1 and HTLV-I. When Mika Popovic, who was leading one of the virology groups in my lab, put that sample in culture, he rapidly detected high levels of reverse transcriptase. And by immunofluorescence, he detected HTLV-I antigens. But then came the conflicting data: Marjorie Robert-Gouroff had run the serology on a large cohort of patients, and HTLV-I positivity was detected only in a minor fraction, less than 5%. What was going on? We started to suspect that the young Frenchman might have been co-infected with HTLV-I and the new agent, and Mika was eventually able to culture the two viruses separately. Although this incident initially gave us a wrong lead, it eventually provided a critical hint: in fact, HTLV-I was immortalizing mature CD4 T cells in those cultures, providing the perfect substrate for growing HIV-1 in large amounts. So, we realized that we needed immortalized T cells to produce the new agent in large quantities. In the end, the young Frenchman’s co-infection turned out to be a blessing.

How important was the role of the clinicians in the discovery of HIV? The clinicians, like Leibowitch and Willy Rozenbaum, deserve a lot of credit because they were the ones who really brought the Pasteur group into action. Willie Rozenbaum, in particular, had the idea to using lymph node tissue instead of blood. If you think about it for a second, you get blood from patients who are dying of AIDS: how many T cells do you expect to find in their blood? Almost none. My lab was receiving only blood samples, and we would get a positive hit of reverse transcriptase in the cultures, which was suggestive of a retrovirus, but then it disappeared right away because there were no CD4 T cells left for virus replication. Some were too low to even measure a blip of reverse transcriptase. So, we had many real short-term isolates, but at that time, none was really characterized. But thanks to Rozenbaum, the Pasteur group started to get lymph node biopsies, and he deserves enormous credit for that. Finally, they got this isolate from the lymph node of a patient named “Bru”, which later turned out not to be from patient “Bru” because even the Pasteur lab, like ours and several others, had their cultures contaminated with LAI/IIIB, a virulent HIV strain that grows like weed. One reason is that IIIB is an X4 strain (i.e., uses CXCR4 as a coreceptor), while the vast majority of wild strains, especially from patients who are not terminally ill, are R5 (i.e., use CCR5 as a coreceptor) and will never grow in regular T-cell lines. Even Luc Montaigner, who had repeatedly denied that he ever had any virus contamination in his lab, had to acknowledge that his cultures got contaminated with LAI/IIIB.

How important was your previous discovery of the first human retrovirus, HTLV-I, and its close relative, HTLV-II? It was fundamental. Without that discovery, nailing down HIV would have been much more challenging because the concept itself of a human retrovirus was extraneous to the scientific community and difficult to accept. A prejudice that we understood way too well when our first paper on HTLV-I was rejected and almost “vilified” by the Journal of Virology. The HTLV-I discovery, which was the result of extraordinary work by some outstanding people in my lab, especially Bernie Poiesz, broke the dogma and showed the world that retroviruses were not restricted to animals and could infect humans as well. Then, of course, the methodology that we developed to discover HTLV-I and -II in the 1970s’ was the same that we applied to the discovery of HIV.

So, you are saying that when you started to look for the cause of AIDS, the technology was already in place. Many young scientists today seem to rely more and more on technology and less and less on creative thinking. How critical was technology in the discovery of HIV? It was critical. But not sufficient. At the beginning of my career, as a young medical doctor, I used to have a lot of insecurity about technology. My Ph.D. colleagues seemed to know everything about technology. I knew too little. So, I was always frustrated and always thought that I needed to learn more. But then you get to realize that technology does not succeed by itself. You cannot make a “golden calf” of technology. You need a mind behind it, to steer it toward the right questions, and to follow the right leads. Choosing the right lead to follow can be hard, especially for the young and inexperienced. You need knowledge, a lot of it, good instinct, and also some luck, that is for sure. However, there were indeed key requirements for the isolation of human retroviruses. The first was the ability to grow primary T cells in vitro. We had solved that problem a few years earlier with the discovery of IL-2, which we called at that time T-cell growth factor, or TCGF. Some call it the first cytokine, even though there was already interferon, which was not considered a cytokine back then. But for sure, it was the first interleukin. This was the basis for it all. And then we needed an assay to identify the footprints of a retrovirus in cell cultures, and this was the reverse transcriptase assay. The seminal discoveries by the late Howard Temin, a dear friend of mine, and David Baltimore had opened the field, but we needed a practical system to detect reverse transcriptase in cell culture supernatants and distinguish it from other confounding polymerase activities present in the cells. And we developed that system, especially thanks to the great work of my colleagues Marv Reitz, Sarang (Mangalasseril Sarangadharan), and Marjorie Robert-Gouroff.

Some reporters have tried to portray the saga around the discovery of HIV as a “war” between the French (Luc Montaigner’s group at Pasteur) and the Americans (Bob Gallo’s group at the NIH), but in reality, it started out as a collaboration across the Atlantic, didn’t it? Of course, it started out as a collaboration between the Pasteur group and us, because initially, I gave the idea of a retrovirus, and then Francoise Barrè-Sinoussi, who at that time was a technician in the laboratory of Jean-Claude Chermann in Montaigner’s Department at Pasteur, traveled to my lab at the NIH to learn how to culture T cells with IL-2 and how to measure 4 Current HIV Research, 2023, Vol. 21, No. 1 Interview reverse transcriptase activity in the cultures. We were glad they were joining us in pursuing the retrovirus hypothesis, and we provided them with all of our protocols and gave them IL-2 and antibodies to HTLV-II and -II. Also, Chermann, who has remained an unsung hero but was the real team leader in the discovery of HIV at Pasteur, was a good friend of mine and visited us many times. And then the first version of their paper on the isolation of a T-lymphotropic retrovirus from a patient at risk for AIDS was rejected at Nature, and I suggested them to submit it to Science, and we helped them to get it published. I was the reviewer at Science and when the Editors argued that there was not enough evidence that their virus was new, I got my colleague Prem Sarin to serve as the second reviewer; we suggested some improvements in the manuscript, and eventually, it got accepted. Later, I was criticized for recommending acceptance in Science because the paper did not provide definitive evidence that their virus was not HTLV-II. But I knew it was the right thing to do. It was urgent to move the field forward as fast as possible. Of course, when I think about it now, it would have been easy to find holes in their paper and reject it, but I am glad we did not. So, we not only collaborated with the French at the beginning of the story, but we also helped them with the publication. Then, unfortunately, came the patent controversy for the blood test revenues, and that created a lot of problems with our collaboration and for them to acknowledge our help. The main limitation of the Pasteur work was that the retrovirus was isolated from an asymptomatic patient, who had just lymphadenopathy but not yet AIDS. So, how could we tell if this virus, even if new, could be the cause of AIDS or simply another opportunistic agent thriving in an immunosuppressed individual? And honestly, from their paper, it was not even so clear that it was a new virus and not merely a variant of HTLV-II, but I knew it had biological effects on T cells that were new. To prove the causal link with AIDS, we needed a blood test, and we needed more isolates to establish a solid linkage to AIDS.

And fortunately, this is the one aspect on which there is no controversy: that you developed the first blood test for HIV, which has saved millions of lives around the world. Well, it is not me again. It is the group. But yes, we decided to go after the blood test right away, because we knew that it was the most urgent thing to do to prove causality and the most consequential to halt the spread of the virus. And we did it.

How did you succeed in such a short time? The breakthrough was the ability to grow the virus in vitro in large quantities. We had outstanding people like Phil Markham and Sarangadharan, but the methods we were using for HTLV-I or -II could not work, because in primary cells, HIV was only growing short-term and did not immortalize T cells. So, Mika Popovic started to throw all the HIV isolates he had in the freezer, we had 48 altogether at that time, into any cell line he could put his hands on, especially T-cell lines, of course. And finally, with my technician Betsy Reed, they found one cell line where the virus started to grow permanently, and that was a Hut-78 clone called H9. In that moment, we knew that in less than 2 months, we would know if this retrovirus was the cause of AIDS. That was the Eureka moment! The tragic irony was that Betsy’s husband was dying of AIDS from a blood transfusion while she was working with the blood of other AIDS patients and helped develop a test that could have saved her husband if only it was available a few months earlier. Once you can grow the virus and you have a lot of it to concentrate, things get much easier to develop an assay. Initially, we started working with ELISA, but soon we found that it was not specific enough: the false positives were too many. And so, we decided that we needed a verification assay to confirm the ELISA positivity, and we thought about Western blot. I had a post-doc from Switzerland in the lab, Jrg Schüpbach, who had worked in a blood bank in Zrich, and he and Sarang started to develop a Western blot, which allows you to see all the proteins by their size. And so, in a few weeks, we were able to back up the ELISA data with a second assay that had a much greater specificity. Previously, Western blot was only a research tool, but we aimed at bringing it to clinical medicine, and we did. At that time, Montaigner’s people were only using immunofluorescence on heterologous lymphocytes derived from different blood donors, which gave those lots of false positives because of the HLA variation between one donor and the other. If you read their first patent, they only had 17% positivity in AIDS patients, which was useless to identify people who were infected but asymptomatic and could transmit the infection.

During your extraordinary career, you made so many seminal contributions that it is difficult to imagine they all came from a single scientist. Do you feel satisfied now or is there something you are still hunting for? I certainly feel much better than in the days when there was all the tension and pressure of HIV discovery and all the controversies, which were really a senseless impediment to science. So, I am happier now: much happier and much more settled. Maybe I am not driven to the same extent as I was back then, but I am certainly as curious as I was back then, and I still feel the sense of wanting to do more. It is not for the competition. Now it is purely for the fun of the science, and I am most interested in solving the pathogenesis of AIDS, which is extremely complex as you know, and I think we are close to it with a series of recent findings that to me are very exciting. You know about the elite controllers, those people who never need therapy because they naturally control HIV in their bodies. Maybe it is only half of a percent of all patients, but we can learn a lot from them. There is a spectrum, right? There are people who die in a much shorter time, like the unlucky lab technician back in those days who was infected while cleaning a centrifuge for mass virus production, got a high load of virus in his blood at the time of infection, and was dead in a few years. That is really fast progression. And then there are regular progressors who develop full-blown AIDS maybe in 8-10 years, and then there are long-term non-progressors, and at the end of the spectrum, elite controllers, who keep the virus down to very low to undetectable levels without treatment. Well, I suspect that the inoculum size that you get at the time of infection counts. Most studies are focused on virus fitness, which some experts say to be lower in these individuals, but if you think about it, the amount of virus you get on day 1 could make a huge difference, even though as far as I am aware, nobody has ever investigated that. In related studies with my long-term colleague and friend, Daniel Zagury, and his Interview Current HIV Research, 2023, Vol. 21, No. 1 5 co-worker, Helène Le Buanec, in Paris, we found that IFN-α plays a critical role in HIV pathogenesis. And then there is another new chapter that we are opening, which relates to a chaperone protein that blocks the ability to repair DNA and completely takes away p53, causing its ubiquitination and presumable degradation, which is also pretty exciting. So, you can see that despite my old age, I am still quite hungry to know more, to learn more, and to discover more.

And finally, Dr. Gallo, is there a lesson for the new generation of scientists that we can take from the HIV discovery saga? I do not know if there is one. The politics were so heavy back then, and the money involved was astronomical. I was told that the HIV patent made more money for the US Treasury than all the other NIH patents in history combined. COVID shares some similarities at the level of financial engagement and the profit for pharmaceutical companies, but the patent war that occurred around HIV affected the academic community very deeply. So, I do not know what to say, if we chop off all the politics and focus only on the scientific endeavor. I am glad that I got an M.D. degree and I am glad that I was insecure about technology because it made me work harder initially, and this paid off later. I would say to the new generation that you have to be daring and never give up. If you get stuck and you are not happy with your research or you are not making progress, do not be afraid to make a move and explore something new. Look at Jim Watson, for example; he left Copenhagen after only one year of post-doc and went to Cambridge where he met Francis Crick who was not that famous, but you know what they have done together. So, do not get stuck if you think you are not making progress, there is always another possibility. You do not want to be trapped with just studying maybe a single segment of heterochromatin for all your life if it is not opening significant new ideas to you. You have to learn the appropriate technology and try to think of something that is important to you and maybe is also important to other people, and then dig in. And you need to have colleagues and friends. Do not isolate yourself. It is important to have time for yourself to think, but you also have to remain engaged with other people. And then try to be humble enough to be ready to learn from other people. There are many kinds of intelligence and you need them all to break through the complexity of reality. You will make mistakes, and I cannot say enough how much I learned from mistakes, but you need to persist. Let me give you an example. Soon after I started at the NIH, I caused a big accident in the lab and I was ready to quit. We were using these humongous 9-feet columns, which had to go through the ceiling of the lab and I had to get permission from the guy above to drill a hole in his floor, which was funny. One day, I was to purify transfer RNA and I got my samples running. It was a nice sunny Saturday afternoon, and I was striving to go out and play tennis, but I was still there hour after hour and this thing was going forever. So, I accelerated the damn pump to make it run faster and faster, and then all of a sudden, boom! It blew up. There I was, with the glass broken and the samples spilled all over the place. So came Monday morning, and I went straight to the lab chief and I said to him: “I am sorry, but I am not made for this. I do not think I am good enough, I am never going to deal with all this technology. I am just too impatient…” And he invited me to his office and there he told me this story: “You know, about 8-9 years ago, we had another M.D., just like you, who came in on a weekend and had to wash the glass pipettes. He set the washer up and left. When we came in on Monday, there was a massive flood in the lab and a huge mess everywhere. So, the young man came to me and told me the same things that you just said: that he did not have it and that he was going to quit. His name was Arthur Kornberg. Fortunately, he did not quit, and a few years later, he won the Nobel Prize for synthesizing DNA in a test tube.” So, he said to me: “Bob, go back to the lab and try again”. I guess it was a good thing that I did not give up.

CONCLUSION
We are grateful to Dr. Gallo for talking with us about his discovery of HIV-1 and especially for his extraordinary contributions to science. Who knows how long it would have taken for him and the Pasteur group to identify HIV-1 without the groundbreaking advances that his laboratory had made in the previous decade. His tenacity and determination in pursuing human retroviruses when only very few people in the field were accepting their mere existence – let alone that they could cause major human diseases – is a great example for young scientists to never give up and to go after their intuitions in spite of adversity and failure. It is fascinating to see how the path to discovery never follows a straight line. There are setbacks, wrong leads, periods of stall, and then, all of a sudden, a “eureka” moment. Brilliance is by and large written in your genes, and Dr. Gallo certainly has multiple copies of those genes. But tenacity and determination are characters that can be acquired. And they are fundamental to make a good scientist.

Read the full interview here: https://bit.ly/3KVHLta

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