Ten years into the making, the HIV-1 mosaic vaccine finally goes into human trial

© Bette Korber et al.

I hope you will all forgive me if this week I'm gushing over my amazing mentor Bette Korber, as last week she shared some awesome news on Facebook:

"A landmark in my life happened yesterday, a major step in a long story. A decade ago I had an idea for making an HIV vaccine that had the potential to work globally. After a struggle (in my first 2 failed proposals, reviewers declared what I proposed was impossible), I got an internal grant from Los Alamos to develop the idea (third time's a charm). With that funding I could bring together a group of computational people to work together on expressing the idea -- a talented guy named Simon Perkins wrote amazing code to make it so, with computational design suggestions from the group, particularly my husband James Theiler. Then James, Will Fischer, Tanmoy Bhattacharya, and I put it through its paces, optimizing running conditions and devising ways to compare mosaics with natural proteins, with additional help from our friends Karina Yusim, Carla Kuiken and Bob Funkhouser. We called it a mosaic vaccine.

After so many years of hard work, and with the collaboration of experimentalists at Harvard and at Duke (Drs. Haynes, Letvin, and Barouch), two weeks ago a phase I safety trial finally opened, and an HIV mosaic vaccine went into the arm of a human volunteer for the very first time. "Safety trial" means that this is just the first phase in testing the safety of the vaccine (I explained the three phases of human trials in this post). We will gather immune responses and we are hoping to see the same good results we saw in monkeys [2-5]. If all goes well, HIV mosaics are in the pipeline for 4 more human vaccine studies. I'm so excited about this study and so proud of my mentor.

When I explain to people the challenge we are facing when designing an HIV-1 vaccine, I usually make a very simplistic comparison with the flu virus. Influenza evolves from one season to the next, which is why every year we need a new flu shot. So, basically, the flu evolves into a new virus every year. Well, HIV evolves so rapidly that every person has a different virus. In our database alone we have half a million distinct HIV viral sequences: how can you vaccinate people against half a million different viruses?

In the past, successful vaccines against diseases like polio or the measles have been made by taking a real virus, inactivating it (for example, you just take one or two of its proteins, but not the whole virus, to ensure it loses its ability to infect cells), and then injecting it into the body. The immune system "sees" the viral proteins and initiates a response. The response is then "saved" into memory cells, which, next time they encounter the pathogen, will remember how to produce the right response that will promptly clear the virus before it can start an active infection.

So, as you can see, the problem with HIV is that the viral population is so diverse that no one virus found in nature will protect people from contracting the infection. How to bypass the obstacle, then? Bette's idea is to basically use a computer that mimics HIV's evolutionary mechanisms to create an in-silico virus [1], something I've discussed in this post. The algorithm takes as input a population of, say, 100 different HIV sequences, and then recombines them creating a new population of artificially constructed viral sequences. HIV viruses can naturally recombine when infecting the same cells, and what the algorithm does is mimic this mechanism making sure that after every recombination step the new sequence is still a viable and functional virus. The computer mimics this process, iterates it multiple times and then the best representative is selected as a potential vaccine.

The first caveat is: is this new, artificially constructed sequence a real virus? After all, it was never found in nature. It was created by a computer algorithm. It turns out that when reconstructed in a wet lab, the mosaic proteins are functional and viable.

The second hurdle was to prove that these artificially constructed sequences are safe to be used in a vaccine and that they do elicit protective responses against not just a few HIV viruses, but many, many HIV viruses -- enough to prevent infection. So, you get an idea of why the mosaic vaccine took 10 years from concept to the first human trial.

Animal studies [2-5] demonstrated that mosaic vaccines elicit good immune responses. In one study in particular [3], compared to controls, vaccinated monkeys required many more challenges to get infected (for a risk reduction of 80%), and once infected, they were able to control the viral load and survive the infection.

So, as Bette said, we are hopeful. Hopeful and excited!

[1] Fischer W, Perkins S, Theiler J, Bhattacharya T, Yusim K, Funkhouser R, Kuiken C, Haynes B, Letvin NL, Walker BD, Hahn BH, & Korber BT (2007). Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants. Nature medicine, 13 (1), 100-6 PMID: 17187074

[2] Nkolola JP, Bricault CA, Cheung A, Shields J, Perry J, Kovacs JM, Giorgi E, van Winsen M, Apetri A, Brinkman-van der Linden EC, Chen B, Korber B, Seaman MS, & Barouch DH (2014). Characterization and immunogenicity of a novel mosaic M HIV-1 gp140 trimer. Journal of virology, 88 (17), 9538-52 PMID: 24965452

[3] Barouch DH, Stephenson KE, Borducchi EN, Smith K, Stanley K, McNally AG, Liu J, Abbink P, Maxfield LF, Seaman MS, Dugast AS, Alter G, Ferguson M, Li W, Earl PL, Moss B, Giorgi EE, Szinger JJ, Eller LA, Billings EA, Rao M, Tovanabutra S, Sanders-Buell E, Weijtens M, Pau MG, Schuitemaker H, Robb ML, Kim JH, Korber BT, & Michael NL (2013). Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV challenges in rhesus monkeys. Cell, 155 (3), 531-9 PMID: 24243013

[4] Santra S, Muldoon M, Watson S, Buzby A, Balachandran H, Carlson KR, Mach L, Kong WP, McKee K, Yang ZY, Rao SS, Mascola JR, Nabel GJ, Korber BT, & Letvin NL (2012). Breadth of cellular and humoral immune responses elicited in rhesus monkeys by multi-valent mosaic and consensus immunogens. Virology, 428 (2), 121-7 PMID: 22521913

[5] Barouch DH, O'Brien KL, Simmons NL, King SL, Abbink P, Maxfield LF, Sun YH, La Porte A, Riggs AM, Lynch DM, Clark SL, Backus K, Perry JR, Seaman MS, Carville A, Mansfield KG, Szinger JJ, Fischer W, Muldoon M, & Korber B (2010). Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys. Nature medicine, 16 (3), 319-23 PMID: 20173752

A new viral vector raises hopes for an HIV vaccine

Because I work on HIV vaccine research, I often talk about vaccines and HIV vaccine design in particular. So far, there have been several phase I HIV vaccine trials, but very few have made it into phase III. One such case was the STEP trial, which was abruptly halted in 2007 after preliminary results showed that not only the vaccine was not protecting people from getting the virus, but the rate of HIV infections was actually higher in the vaccinated subjects compared to the subjects that received a placebo. Even more alarming was that this increase in acquisition risk lasted years after vaccination.

What went wrong in the STEP trial?

Vaccines are made of a "wimpy" version of the virus: you have to use enough genetic material from the virus in order to induce antibody production, but not enough to start an infection. We call the modified virus used in a vaccine "immunogen." The immunogen is only one part of the vaccine "recipe", the other part is what we call a vector, a structure that carries the immunogen and presents it to the immune system. Viruses make excellent vectors because they are like little "boxes" that are programmed to enter cells. And of all possible viral vectors, the most often used are adenoviruses because they are very common in the human population (they cause the common cold) and are therefore considered to be safe to "hijack" into carrying vaccine immunogens.

The STEP HIV vaccine was made of an adenovirus vector (recombinant adenovirus serotype 5 or rAd5) expressing the HIV proteins gag, net, and pol. When researchers looked back at what could've possibly gone wrong they found that the rates of infections were significantly higher in subjects that had been previously infected with Ad5 and had preexisting immunity against Ad5.

The HIV community feels so baffled by the failure of the STEP vaccine trial that at a recent conference I attended, the director of the Fred Hutchinson Cancer Research Center said quite vehemently that we should all move away from vector vaccines and do DNA vaccines instead. Since DNA is naturally absorbed by cells, DNA vaccines bypass the need of a vector.

In truth there's still strong hopes for vector vaccines. The natural question to ask in light of what happened with the STEP trial is: can we use a vector that instead of worsening the immune response actually makes it better?

It turns out that there is, and it's called Cytomegalovirus, or CMV. Like adenoviruses, CMV's are also very common in the human population and typically asymptomatic unless there are other underlying conditions.

If you remember roughly how the immune system works, we have two kinds of "sentinels" looking out for invaders: B-cells, which produce antibodies, and T-cells. While antibodies bind to viral particles, thus preventing the virus to enter and infect cells, T-cells recognize infected cells and destroy them. This recognition mechanism is based on the fact that infected cells express fragments of viral proteins (epitopes) on their surface. The T-cell recognizes those proteins as foreign and as a flag of infection and thus kill the cell before it starts replicating the virus.

Eliciting antibodies able to clear the HIV virus through a vaccine has proven very challenging (I discuss why in this post). But what about T-cell vaccines? In [2] Hensen et al. showed that a CMV vector SIV vaccine was able to elicit over three times greater breadth T-cell response in rhesus monkeys and about 50% of the vaccinated animals, once challenged with SIV (the simian version of HIV) were able to clear the infection without getting sick.

The vaccine was made of a recombinant rhesus monkey cytomegalovirus (strain 68-1 RhCMV) engineered to express simian immunodeficiency virus (SIV) genes.

"The key finding of Hansen et al. is that strain 68-1 RhCMV elicited CD8+ T cell responses that target SIV epitopes that were completely different from those generated by SIV infection itself, by other virus-based vectors, or by wild-type RhCMV expressing SIV genes [1]."

Typically during an HIV infection, the immune system starts producing T-cells that attack a limited number of epitopes, in other words a limited number of viral protein fragments that infected cells express on their surface. So, the key finding in this study was that using a CMV vector increased the number and variety of epitopes that the T-cells were able to recognize.

"We conclude that RhCMV has an intrinsic ability to elicit CD8+ T cell responses to unconventional epitopes, distinct in quality and quantity from all infectious agents studied to date. [2]."

As you know, HIV's winning strategy to evade the immune system is its ability to "hide" by constantly changing its genetic structure. This is favored by the fact that under normal circumstances T-cells recognize only a limited number of epitopes. In this light you can see why increasing the magnitude and breadth of the T-cell responses is effective in defeating the virus: once primed with the CMV vector, T-cells were not only able to recognize many more epitopes, but different "versions" of such epitopes, meaning that even when the virus came up with a mutation at a certain epitope, the T-cells were still able to recognize it and kill the infected cell.

These are remarkable results and I can't wait to follow this story as it moves to its next step -- human clinical trials.

[1] Nilu Goonetilleke, Andrew J. McMichael (2013). Antigen Processing Takes a New Direction Science DOI: 10.1126/science.1239649

[2] Scott G. Hansen, Jonah B. Sacha, Colette M. Hughes, Julia C. Ford, Benjamin J. Burwitz, Isabel Scholz, Roxanne M. Gilbride, Matthew S. Lewis, Awbrey N. Gilliam, Abigail B. Ventura, Daniel Malouli, Guangwu Xu, Rebecca Richards, Nathan Whizin, Jason S. Reed (2013). Cytomegalovirus Vectors Violate CD8+ T Cell Epitope Recognition Paradigms Science DOI: 10.1126/science.1237874

Vaccines: what is the meaning of phase I, II and III?

I'm often asked, "How long will it take to finally have an HIV vaccine? Are we close? What about this study that published good results on an HIV vaccine?"

Right now, the HIV community is generally optimistic that we will indeed have an HIV vaccine within the next decade. This is based on the relatively recent discovery of new broadly neutralizing antibodies and the mildly positive results obtained by one of the five major efficacy trials, the RV144 Thai trial, which found a 31% reduction in HIV acquisition in vaccinated subjects versus placebo [1].

I'm also often forwarded published papers on successful HIV vaccine trials, with the attached question: "Is it done, then?"

The answer is, "No, not yet."

As I explained in my earlier HPV vaccine post, once a vaccine is approved to be tested on humans, like all human health interventions, it has to be tested in three phase clinical trials, called phase I, II, and III.

"Clinical product development typically begins with phase I studies that evaluate the safety and biological activity of a drug, vaccine, or other intervention and proceeds ultimately to phase III efficacy trials that support licensure. [. . .] Phase II clinical trial evaluation affords an opportunity to discover less frequent side effects of the intervention and to provide better quantitation of the agent‚ activity and safety in a larger and more diverse participant population. [2]."

So, a successful phase I trial means that the vaccine is safe to use on humans and it does no harm. A phase I trial does not prove that the vaccine can protect against the disease. It can take up to a decade to go from a phase I to a phase III trial. Phase III, when successful, is what ultimately proves the vaccine's efficacy.

So far there have been many phase I HIV vaccine trials, but there only have been a handful phase III trials, of which the most successful one was RV144 with the mild 31% reduction in infection rate.

Vaccines like HPV that are now being offered to the public have undergone all three clinical trial phases. This is what I was trying to explain when I discussed the HPV vaccine and I said that despite the concerns raised by the Japanese government, the vaccine wouldn't have been FDA approved had it not passed all three phases of clinical trials that proved its safety first. For example, you can read the results of a phase I HPV vaccine trial here. Notice that the paper was published in 2000 and it took roughly another decade before the vaccine was distributed.

"Before the question of drug or vaccine efficacy can be answered, safety testing, validation of mechanism, and specificity issues must be addressed in preliminary studies. These studies themselves often provide unexpected information that generates new hypotheses. An efficacy trial, usually a randomized controlled trial‚ represents the ultimate test of concept that an intervention can ameliorate disease or prevent infection [2]."

Phase I and II trials are also important for hypothesis raising, not just hypothesis testing. Back to the HIV example, we still don't know why it takes so long for the human body to produce antibodies able to recognize a broad spectrum of HIV strains. We still don't know why a small percent of HIV-infected subjects, the so called "elite-controllers", are able to keep their viral load down to undetectable for decades. We still don't have biomarkers that predict the strength of an immunological response to the vaccine. People who make strong antibodies, they make them later in the infections, when it's too late to clear the virus. Elite controllers, on the other hand, have very low antibody titers.

Finally, to make things even more complicated, the animal models used to test vaccines are not good predictors of the human immune system. For examples, vaccinated macaques have been challenged with SIV, the simian immunodeficiency virus, which is a much older virus than HIV. There are ways to "humanize" the monkeys, but they can never 100% predict the human trial. And that's why we've been eagerly waiting for phase I of the mosaic vaccine... unfortunately, we are still waiting. I should have an update soon, though, as I'm heading out to see our collaborators later this week. Stay tuned!

[1] Supachai Rerks-Ngarm, et al. (2009). Vaccination with ALVAC and AIDSVAX to Prevent HIV-1 Infection in Thailand N Engl J Med DOI: 10.1783/147118910790291082

[2] Lawrence Corey, Gary J. Nabel, Carl Dieffenbach, Peter Gilbert, Barton F. Haynes, Margaret Johnston, James Kublin, H. Clifford Lane, Giuseppe Pantaleo, Louis J. Picker and Anthony S. Fauci (2011). HIV-1 Vaccines and Adaptive Trial Designs Sci Transl Med DOI: 10.1126/scitranslmed.3001863

The virus-antibody arms race

One of the new concepts I learned when I started working on HIV was the most recent common ancestor, or MRCA. When you look at the genetic make-up of a population, you will find a certain amount of variety but also a much greater amount of overlap, i.e. stretches of DNA that are identical throughout the population. Using phylogenetics, one can look at these patterns of shared vs. mutated stretches, and reconstruct the genetic ancestor of the population. For example, you've probably heard of Mitochondrial Eve: since we all inherit our mitochndrial DNA from our mothers, scientists have been able to look at the mitochondrial DNA across all populations and determine the one ancestor (our common mother, so to speak) from which they all originated. Pretty cool, right?

My line of work, for the past 6-7 years has been estimating most common recent ancestors, or MRCAs, of HIV-1 populations. A few years ago we found that in sexually transmitted infections only a handful of viruses are able to come across the genital mucosa and start the infection. Therefore, if you draw a blood sample early enough (a few weeks) after the start of the infection, from that sample we can infer the MRCA of the viral population in the patient. This is particularly relevant because in the case of a viral infection, the MRCA is likely to be the virus that initiated the infection. As the infection progresses, the viral population changes, but it is the ones that are able to break the mucosal barrier (i.e. the MRCAs) that a vaccine needs to target.

Once inside the host, viral evolution is (for the most part) driven by the host's immune system as it tries to counter-attack the infection. At the same time, as the virus changes its genetic make-up to escape the immune pressure, the immune system itself changes and tries to come up with new ways to neutralize the enemy. It's an arms race that in HIV infections typically sees the immune system always one step behind: the first antibodies found in an HIV-1 infected person react with the first, unmutated virus that initiated the infection (the MRCA). As the infection progresses and the virus evolves, new antibodies are made that are able to react to the following viral generations, but typically there's always a subpolulation of viruses that's one step ahead of the antibodies and can still escape. (I hope this part is clear, I've been struggling quite a bit to find the right wording for this paragraph, so if it's not clear feel free to ask questions in the comments.)

In order to design an efficient vaccine, we need to find a way to elicit broad neutralizing antibodies, where by "broad" we mean antibodies that react not only to the present or past viral generations in one host, but to a wide variety of viruses across different hosts and populations. Such antibodies are found in a minority of HIV-infected patients and, typically, by the time they arise, the infection is so spread that they cannot clear the virus.

Ideally, a vaccine should boost a "short-cut" in the evolutionary path that leads to the production of broadly neutralizing antibodies much faster than our bodies are currently capable of. Unfortunately, all vaccine trials attempted so far have not been able to elicit broad neutralizing antibodies. Why?

Antibodies are made by B-cells, white blood cells produced in the bone marrow. In order to produce antibodies, B cells need to be activated, which happens when they find an antigen specific to their receptor. Once activated, B cells not only start producing antibodies, but they also either become memory cells (so that if the antigen is encountered again, the immune system will know which antibodies to produce in order to clear it) or they undergo further differentiation. This process of undergoing more differentiations ensures that the "match" between receptor and antigen becomes tighter and tighter. It takes many cycles of differentiations to produce HIV-1 broadly neutralizing antibodies, and, currently, the process takes so long that most patients don't produce them ever, and the ones that do, don't get them in time to clear the infection.

One reason why we believe it takes many differentiations to make HIV broadly neutralizing antibodies is that they share many similarities to self-reacting antibodies, antibodies that are normally destroyed by the body because they carry a high risk to originate auto-immune disorders (when the immune system attacks its own self instead of antigens). So, instead of eliciting the actual antibodies, could a vaccine elicit its ancestor? Remember how I said that the viral population constantly evolves and, hand in hand, so do the antibodies? Since we can estimate the viral ancestors, can we do the same for the antibodies? Can we reconstruct the differentiation pathway that leads to broadly neutralizing antibodies?

In [1], Liao and colleagues have reconstructed the lineage of the infecting virus in one African HIV-infected patient (CH505), as well as the lineage of an antibody, found in the same patient, able to neutralize 55% of ~200 HIV-1 isolates. the researchers effectively reconstructed the coevolution of virus and antibody within the patient. The patient was followed from week 6 after the infection up until 236 weeks after the infection, and during this period no antiretroviral therapy was administered. This is important because it means that the viral evolution was driven solely by the immune pressure.

Liao et al. found that the first unmutated ancestor in the B-cell lineage appears at week 14 after the infection, and it keeps mutating in ways that are reflected in the evolution of the virus. Once they retraced all the intermediate steps that led to the production of the broadly neutralizing antibody, the researchers tested all of the intermediate antibodies for reactivity against the virus, from the infecting strain to its later generations. They found that breadth and strength of reactivity increased as the antibody lineage evolved. In light of what I tried to explain above, this is a fantastic step forward in understanding how the virus evolves under the immune pressure, as it can help design a vaccine that elicits antibodies that are one step ahead (instead of behind) in the virus-host arms race.

"Thus, a candidate vaccine concept could be to use the CH505 transmitted/founder Env or Env subunits (to avoid dominant Env non-neutralizing epitopes) to initially activate an appropriate naive B-cell response, followed by boosting with subsequently evolved CH505 Env variants either given in combination, to mimic the high diversity observed in vivo during affinity maturation, or in series, using vaccine immunogens specifically selected to trigger the appropriate maturation pathway by high-affinity binding to the unmutated common ancestor and antibody intermediates. [. . .] The finding that the transmitted/founder Env can be the stimulator of a potent BnAb and bind optimally to that broadly neutralizing antibody unmutated common ancestor is a crucial insight for vaccine design, and could allow the induction of broadly neutralizing antibodies by targeting unmutated common ancestors and intermediate ancestors of broadly neutralizing antibody clonal lineage trees."

Of course, there's the usual caveats: will this kind of pathway be reproducible in other patients? How much of it is randomness and how much is it not only retraceable but reproducible is something we will only understand by getting more data from more patients. But it's a start, and a very promising one.

[1] Liao, H., Lynch, R., Zhou, T., Gao, F., Alam, S., Boyd, S., Fire, A., Roskin, K., Schramm, C., Zhang, Z., Zhu, J., Shapiro, L., Becker, J., Benjamin, B., Blakesley, R., Bouffard, G., Brooks, S., Coleman, H., Dekhtyar, M., Gregory, M., Guan, X., Gupta, J., Han, J., Hargrove, A., Ho, S., Johnson, T., Legaspi, R., Lovett, S., Maduro, Q., Masiello, C., Maskeri, B., McDowell, J., Montemayor, C., Mullikin, J., Park, M., Riebow, N., Schandler, K., Schmidt, B., Sison, C., Stantripop, M., Thomas, J., Thomas, P., Vemulapalli, M., Young, A., Mullikin, J., Gnanakaran, S., Hraber, P., Wiehe, K., Kelsoe, G., Yang, G., Xia, S., Montefiori, D., Parks, R., Lloyd, K., Scearce, R., Soderberg, K., Cohen, M., Kamanga, G., Louder, M., Tran, L., Chen, Y., Cai, F., Chen, S., Moquin, S., Du, X., Joyce, M., Srivatsan, S., Zhang, B., Zheng, A., Shaw, G., Hahn, B., Kepler, T., Korber, B., Kwong, P., Mascola, J., & Haynes, B. (2013). Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus Nature, 496 (7446), 469-476 DOI: 10.1038/nature12053

Can we functionally cure HIV?

Last March, Dr. Deborah Persaud, from the John's Hopkins Children Center, presented a stunning finding at the conference CROI, receiving great resonance across several newscasts: Persaud reported the first case of infant functionally cured of HIV. You can watch Persaud's presentation by downloading the podcast here, it's the seventh talk of the session "Is there hope for HIV eradication?"

Up until this finding, the only living person cured from HIV was the Berlin Patient, who was cured after receiving gene therapy for his underlying leukemia condition. Despite this one successful case, gene therapy is not a feasible way to cure HIV.

What does it mean to be functionally cured?

Once in the host, the HIV virus establishes reservoirs of latent virus: these are viral particles that stay dormant in cells and tissues and have the ability to quickly rebound in the event that therapy is discontinued. That's why it's so important for an HIV infected person to never discontinue the drug regimen, as the rebound virus may be drug resistant. HIV is so efficient at escaping the immune system and therapy that standard practice these days is a lifetime of not just one, but a cocktail of 3-4 antiretroviral drugs.

To be functionally cured means that drugs are no longer needed to keep the viral load (amount of virus in the blood) in check (close or below detection), something that until now had only been achieved by an extremely low number of HIV-positive individuals (less than 1% of infected adults), the so-called "elite controllers." In all other subjects, the reservoirs are never completely weakened and they enable the virus to bounce back once therapy is interrupted.

So, what was different with this child?

The mother went into labor without prenatal care. An HIV test was done during labor and normally, when the test is positive, antiretroviral drugs are administered. This is highly effective in preventing mother-to-infant infections as the only moment when the infant is exposed to the mother's blood is at birth. The antiretroviral drugs keep the viral load so low that the risk of infection becomes very small (around 2%). Unfortunately, in this particular case, the birth was so precipitous that there was no time to administer such drugs. The newborn baby was immediately tested for HIV.

This is my understanding of what was unique about this case: normally a first test is done and, if positive, a second follow-up test is performed and prophylaxis is started once the infection is confirmed. In this case, though, two independent tests were done at the same time and, since both confirmed the HIV infection, prophylactic treatment was started very early, when the baby was 31 hours of age. Also, unique to this case was the fact that a regimen of three drugs, of which one at the therapeutic level instead of the standard prophylactic dosage, was administered during the first week of life. After that, the baby was switched to a standard treatment of antiretroviral drugs (again, my understanding from the CROI talk).

Such regimen successfully brought the child's viral load down to undetectable, which is normal in these cases. Despite this, because of HIV's ability to establish reservoirs, antiretroviral therapy is never discontinued. Like I said before, it is a lifetime therapy. So called "drug holidays" result in more virulent and drug-resistant HIV quasispecies. However, this child was lost to follow-up at 18 months of age and was once again seen by the doctors at 25 months of age, when the caregiver reported discontinuing the therapy. Immediate testing was done to assess the child's viral loads. The child was tested not once, but many times. Genetic testing was also done to make sure it was the same child treated before. The doctors must have been in disbelief as for the first time they were seeing the incredible: after 5 months since discontinuing antiretroviral therapy, the viral load in this child was still undetectable.

What are the consequences? As Dr. Persaud repeated many times during her talk, this is a single case and a proof of concept. We need more cases to be able to generalize (as statistics teach us). However, it points to something that indeed needs to be explored: how early in the infection can we (and should we) intervene? In a 2012 paper [1], Persaud and colleagues studied the dynamics of the latent HIV reservoirs in 17 infants on very early antiretroviral drug therapy (median start age 8 weeks) and found that the size of the reservoirs at age 2 was associated to how early undetectable viral loads were achieved during therapy. The earlier viral load was suppressed through therapy, the smaller the HIV reservoir at age 2. Is there a point, very early into the infection, when the virus is vulnerable and all reservoirs can be not just reduced in size, but actually completely eradicated through potent and prompt intervention?

In rare cases, HIV-infected patients are able to spontaneously maintain their viral load at a very low level without the need of drugs, the so called "elite controllers." What if, when administered early enough, antiretroviral drugs could transfer this type of spontaneous protection to every HIV-infected person?

Shortly after the CROI conference, a French study published in PLoS Pathogens [2] reported 14 cases of what they call "post-treatment controllers," in other words, people whose viral loads remained very low after interrupting treatment. With the exception of mother-to-infant transmissions at birth, it's extremely hard to catch this virus early because people often don't realize they've been infected: symptoms, if any, appear 3-4 weeks later and are often mistaken for a common cold. Twelve of the 14 cases reported in [2] had symptoms that prompted early intervention and start of therapy during the primary infection.

"Post-treatment controllers (PTCs) had a more severe primary infection with higher viral loads and were frequently symptomatic, which may have prompted the early treatment in some cases [. . .] Therefore, our results strongly suggest that the infection control in the PTCs was not achieved spontaneously and was favored by the early onset of therapy. Because the interruption of long-term antiretroviral therapy initiated early during primary infection is not recommended, only a very small proportion (~2%) of the patients in the French Hospital Database on HIV Infection experienced such an interruption, which may explain the rarity of PTCs worldwide [2]."

[1] Persaud, D., Palumbo, P., Ziemniak, C., Hughes, M., Alvero, C., Luzuriaga, K., Yogev, R., Capparelli, E., & Chadwick, E. (2012). Dynamics of the resting CD4+ T-cell latent HIV reservoir in infants initiating HAART less than 6 months of age AIDS, 26 (12), 1483-1490 DOI: 10.1097/QAD.0b013e3283553638

[2] Sáez-Cirión, A., Bacchus, C., Hocqueloux, L., Avettand-Fenoel, V., Girault, I., Lecuroux, C., Potard, V., Versmisse, P., Melard, A., Prazuck, T., Descours, B., Guergnon, J., Viard, J., Boufassa, F., Lambotte, O., Goujard, C., Meyer, L., Costagliola, D., Venet, A., Pancino, G., Autran, B., Rouzioux, C., & , . (2013). Post-Treatment HIV-1 Controllers with a Long-Term Virological Remission after the Interruption of Early Initiated Antiretroviral Therapy ANRS VISCONTI Study PLoS Pathogens, 9 (3) DOI: 10.1371/journal.ppat.1003211

Make a donation to NOAH, get a free 8x12 print

As many of you know, my research is on HIV, with a focus on HIV vaccine design. I work in Bette Korber's group, and through Bette, I came to learn about NOAH, an organization she co-founded.

Taking care of AIDS orphans has been one of the most prominent issues in Sub-Saharan Africa, where two-thirds of the people affected by HIV/AIDS live. Mother-to-infant transmissions are highly preventable yet, sadly, the drugs are expensive and not always available in Africa. A staggering 2.5 million African children have been orphaned by AIDS, and many of them are born HIV-positive. There are villages where a whole generation has disappeared because of AIDS.

NOAH takes care of these children without taking them away from their homes. NOAH is not an orphanage. The organization provides schooling, day care and food for the kids, while the kids continue to live in their village with older relatives. $80 covers one child for one year.

For the whole month of December, if you make a donation of $30 or more, I will send you an 8x12 print of one of my pictures. Follow this link to donate, forward the email receipt to eegiorgi (at) gmail.com, include your shipping address, and pick the picture of your choice from my G+ album (click on "Photo details" to see the file name).

Donations are tax-deductible.

THANK YOU!

The beginning of the end. . . Maybe.

"We share a very special moment - it is the moment when an AIDS-free generation is finally in sight." That's what the US president, Barack Obama, said on July 26.

Well, are we?

A colleague a few days ago brought to our attention some stunning figures: according to the CDC, of all HIV infected individuals in the US, only 25% are under treatment and hence have the virus under control. Quite striking if you consider that in Sweden instead 85% of HIV-positive individuals are undergoing treatment. The consequences of such a poor statistic in the US go beyond the lifespan of the single individual: people under antiretroviral therapy have much lower viral loads and therefore a significantly reduced chance of passing the virus to their partners.

I'm not surprised by the CDC numbers, actually. When newly infected, subjects have no symptoms or may feel like they are coming down with the flu. You can live with this virus for ten years without having symptoms. If you don't have health insurance, and you are feeling well, why bother go see a doctor? In the meantime, though, these individuals continue to spread the virus. And the problem doesn't affect the US alone: according to the World Health Organization, less than half of the infected people worldwide are actually receiving treatment.

These were my thoughts as I read the perspective article "The beginning of the end of AIDS?" in NEJM [1]. The authors base their cautious optimism on a few things: mildly positive results on a recent vaccine trial, more effective drugs, and the news of the first patient to ever be cured of HIV. The latter I discussed in this post. The news was indeed exceptional but, unfortunately, gene therapy is not the way to stop this pandemic: 2/3 of people currently living with HIV/AIDS are in sub-saharan Africa, where drugs are still hard to find, let alone extremely costly procedures like gene therapy. And more effective drugs are not going to solve the problem if they remain unaffordable or unavailable to the majority of infected people.

So yes, in the end, it all boils down to funding:

"Global resources have been declining, not growing, in this period of scientific success. This lack of funding is the major point of divergence between optimism and pessimism."

Why invest on HIV?

"Comprehensive economic models predict that making the needed investments in HIV-related efforts will result in cost savings over the long term."

HIV debilitates the immune system. The effects of diseases like tuberculosis, hepatitis and malaria could be reduced if the spread of HIV could be reversed because of the effect the virus has on the immune system. Making antiretroviral treatment available to all infected people is the best strategy: by keeping the viral load under control, drugs effectively lower the rates of mother-to-infant and sexual infections. According to the CDC, about 100-200 infants are born every year in the US with the virus in their body. Adequate treatment during pregnancy and delivery can reduce the rate of mother-to-child transmission to less than 2%.

As we strive to reach out to every infected person on the planet, funding must not stop for research. A vaccine is the most affordable and most effective way to stop the pandemic and we have to keep pushing in that direction. My supervisor gave a talk last week in which she outlined where we are in terms of vaccine research. She opened the talk remembering how the first vaccine was discovered: the English physician Edward Jenner (1749 - 1823) rubbed pus collected from blisters milkmaids received from cowpox on his gardner's eight-year-old son. He then exposed the boy to pox, twice, and noticed that they boy didn't develop the disease. The audience was of course horrified when my supervisor mentioned the sacrifice of the little boy, and yet when she went on describing how long and, most importantly, how much money is needed to develop and test a vaccine, a girl in the audience raised her hand and asked: "Well, maybe you can't take an eight-year-old boy, but wouldn't you be better off testing the vaccine on yourself?"

We can't, of course. The gardner's boy got lucky, but things don't always go well. The field still hasn't forgotten the failure of the Merck vaccine in 2007, the trial that was halted after the experimental vaccine was found to make some subjects more susceptible to infection. The FDA has a set of very strict regulations on vaccines. They need to be stable, in other words, one has to show that they don't change after they've been grown for several generations in cultures. They have to be attenuated, and remain so after several generations. Vaccines are then tested on mice, first, then monkeys, then, years later, on humans in several phases that take time, money, and then more time and more money. And yet what we really cannot afford is to stop pushing the research forward.

"Every country must develop more effective ways to reach key affected populations and to apply the tools that we know work, if we are to make significant advances."

So, Mr. President, I hope you are right. But I also hope you will keep funding our efforts.

[1] Diane Havlir, & Chris Beyrer (2012). The Beginning of the End of AIDS? New England Journal of Medicine : 10.1056/NEJMp1207138

Hijacking dendritic cells

Dendritic cells are antigen-presenting cells: their main function is to patrol in search for "foreign objects" (the antigens). When it finds an antigen, the dendritic cell "chops it up" in fragments that are then presented to its surface. In more technical terms, it takes up the antigen by either phagocytosis or receptor-mediated endocytosis and transfers it to the cytosol, where further degradation may occur via proteosome. From here the resulting antigen peptides enter the endoplasmic reticulum and, ultimately, are exposed on the cell surface. At this point the dendritic cell migrates to the lymph nodes, which are rich in T-cells. The antigen fragments it carries on its surface are like red flags: once a T-cell recognizes a specific fragment, it gets "activated" and new T-cells with the same antigen specificity are created in order to mount an immune response against the invader.

Now, as you know, HIV infects preferentially T-cells. However, as a sexually transmitted virus, it first enters the body through the genital mucosa. How does the virus find T-cells from there? Easy. It highjacks dendritic cells and takes a ride to the lymph nodes, where the T-cells are.

But wait... the dendritic cell is supposed to kill the virus, not give it a ride...

Unfortunately, HIV has developed a mechanism that allows it to escape the usual degradation process inside the dendritic cell. It is capable of hitchhiking the dendritic cell without compromising its infectivity by residing "in an invaginated domain within the cells that is both contiguous with the plasma membrane and distinct from classical endocytic vesicles [1]." These are small membrane vesicles that are referred to as exosomes. They may be released in the extracellular milieu, following fusion of the multivesicular bodies with the plasma membrane. Once inside the dendritic cell, the virus can infect a T-cell via a mechanism called trans-infection, where the virus is passed from one cell to the other through the release/fusion mechanism of the exosome.

As you can imagine, these mechanisms are very interesting to study because if we could block the "highjacking" of the dendritic cells at the mucosa level, we could possibly stop the virus from spreading to the T-cells and initiate the infection. A team of researchers from Spain and Germany have studied this mechanism extensively. In a 2010 paper [1], Izquierdo-Useros et al. suggested that mature dendritic cell trans-infection could play an important role in augmenting "viral dissemination in the lymphoid tissue and significantly contribute to HIV disease progression." Mature dendritic cell encounter many T-cells every hour, with contacts that last several minutes, and as a consequence they have the potential to infect a broad number of T-cells. This could explain why HIV productive infection is more likely in subjects with a pre-existing sexual infection: the pre-existing mucosal inflammation could be responsible for the mobilization of a higher number of dendritic cells, which, in turn, could favor the spread of the HIV virus.

In a paper published last month [2], the research team showed that a particular class of lipids on the HIV surface favors the uptake of the virus into the dendritic cells. These lipids, called gangliosides, are a group of glycosphingolipids that are comprised of a ceramide linked to several oligosaccharide chains. They are basic components of the host cell’s plasma membrane, and they get incorporated into the viral envelope (the outer shell of the virus) when a new viral particles buds out of the cell. Izquierdo-Useros et al. [2] used artificial virus-like particle to show that only viruses with these lipids present on their surface were able to get into the dendritic cells.

Together, these findings pave new ways for novel strategies to block the spread of the HIV virus in the body, as well as a possible dendritic cell based vaccine.

Edit: As I was browsing the latest PNAS issue, I noticed an independent paper that reports the same finding that glycosphingolipid GM3 on the HIV-1 envelope allows for viral capture by mature dendritic cells. I've included the citation below [3].

[1] Izquierdo-Useros, N., Naranjo-Gómez, M., Erkizia, I., Puertas, M., Borràs, F., Blanco, J., & Martinez-Picado, J. (2010). HIV and Mature Dendritic Cells: Trojan Exosomes Riding the Trojan Horse? PLoS Pathogens, 6 (3) DOI: 10.1371/journal.ppat.1000740

[2] Izquierdo-Useros, N., Lorizate, M., Contreras, F., Rodriguez-Plata, M., Glass, B., Erkizia, I., Prado, J., Casas, J., Fabriàs, G., Kräusslich, H., & Martinez-Picado, J. (2012). Sialyllactose in Viral Membrane Gangliosides Is a Novel Molecular Recognition Pattern for Mature Dendritic Cell Capture of HIV-1 PLoS Biology, 10 (4) DOI: 10.1371/journal.pbio.1001315.

[3] Puryear, W., Yu, X., Ramirez, N., Reinhard, B., & Gummuluru, S. (2012). HIV-1 incorporation of host-cell-derived glycosphingolipid GM3 allows for capture by mature dendritic cells Proceedings of the National Academy of Sciences, 109 (19), 7475-7480 DOI: 10.1073/pnas.1201104109
 

Different viruses, different replication mechanisms

I often talk about HIV because that's my research field. However, HIV is not the only virus for which we currently don't have a vaccine. A recent news post on Scientific American warned that while HIV-related deaths are going down, chronic hepatitis C deaths are on the rise. According to the World Health Organization:

"It is estimated that 3-4 million people are infected with HCV each year. Some 130-170 million people are chronically infected with HCV and at risk of developing liver cirrhosis and/or liver cancer. More than 350,000 people die from HCV-related liver diseases each year."

HCV has many similarities with HIV: they are both highly variable with comparable mutation rates, leading to multiple subtypes, and they are both RNA viruses with similarly sized genomes (~9,500 bases). They also have similar clinical patterns, as the acute phase (first few months) is either asymptomatic or very mild in both infections, and is characterized by a rapid ramp-up in viral load (levels of viral RNA per blood unit). However, while the HIV viral load, after reaching a peak, comes back down, the HCV viral load reaches a plateau and remains constant for many weeks. Eventually, only 20-30% of individuals infected with hepatitis C will spontaneously clear the virus and resolve the infection, whereas 70-80% will progress to persistent infection.

The biology of the two viruses is also very different, and if, like me, you thought RNA viruses are all alike, think again.

A virus needs to use the host cell machinery in order to reproduce. Different viruses have developed different mechanisms in order to do this. The key step is to use the genetic information they carry in order to make new viral proteins and hence new viral progeny.

Retroviruses are RNA viruses that, in order to produce new progeny, they need the DNA intermediate step. HIV is one of such viruses: once inside the cell, its RNA is transcribed into DNA by an enzyme called reverse transcriptase (also packaged inside the virion). The viral genome is then transported to the cell nucleus by another enzyme and is integrated into the host genome. This is a fundamental step for HIV, because it allows it to utilize the cell machinery in order to reproduce itself.

HCV also uses the cell machinery, but in a different way. Instead of using reverse transcriptase to turn the RNA into DNA, it uses a different enzyme, called RNA polymerase, which produces messenger RNA from which viral proteins are made. Through this step, HCV makes new negative RNA strands that serve as templates for the new progeny. The negative RNA templates stay inside the cell and continue to produce positive RNAs, while the positive strands may either be used to produce a new negative strand template, or they may be packaged into new virions, or they may be translated into proteins. All of this happens in the cytoplasm, and, contrary to HIV, HCV never enters the cell nucleus. HCV replication and post-translational processing happen in a "membranous web" called "replication complex," and then new virions are matured in the Golgi apparatus before being released outside the cell through exocytosis.

So, you see, though all retroviruses are RNA viruses, the opposite is not true. I learned something new today. Thank goodness I learned it in time for my talk on Thursday!

Ashfaq, U., Javed, T., Rehman, S., Nawaz, Z., & Riazuddin, S. (2011). An overview of HCV molecular biology, replication and immune responses Virology Journal, 8 (1) DOI: 10.1186/1743-422X-8-161

Human Immunodeficiency Virus model

Human Immunodeficiency Virus model

This is so darn cool! Well, okay, for an HIV scientist like me... But still, click on the link: it'll give you all the inside views of the virus, and then, if you click on "view labels" on the upper left corner, it tells you what you're looking at and the reference to the paper that describes it. So awesome to have it all conveniently in one picture!

GP 120!

I took the above photo yesterday. As I was processing it, I suddenly froze and thought, "GP 120!"

Check it out:

GP120 is the protein that sits on the outer shell (envelope) of HIV and binds to target cells. A trimer of three gp120 (together with three gp41, another protein) bound together forms the "spike" you see in the above picture.

Something tells me I've been working too hard if I suddenly see gp120 in tulips.
Fascinating, though, how these trimeric structures come up in nature, don't you think?

Mapping HIV-human protein to protein interaction reveals new targets for better drug design

HIV has a small genome (roughly 9,000 bases) and it survives by using the host's proteins and DNA. Understanding how these proteins come in contact and interact with one another is crucial in order to unravel the mechanisms by which HIV hijacks the cellular machinery and proliferates. A comprehensive work [1] by a group of researchers at UCSF lead by Nevan Krogan looked at two human cell lines in particular and identified 497 HIV-human protein-protein interactions between 16 HIV proteins and 435 human factors. The study, published in the last issue of Nature, is the first one to look at protein-protein interactions in a host-pathogen system, and it opens up new possible targets for drug design.

The researchers devised a score to classify the strength of the interactions, which they statistically validated through random reshuffling. They identified 196 interactions in both cell types, while 150 and 151 were specific to each line (HEK293 and Jurkat cells respectively, two human cell lines that were isolated in the '70s and are used today in experiments). Interestingly, the proteins identified in both cell lines had stronger evolutionary signatures than the others, something the researchers were able to identify using comparative genomics between human and rhesus macaque.

Besides revealing an enrichment for host proteins that the virus recruits in order to replicate, the study unveiled proteins that have an inhibitory role during the infection. For example, they knocked down ten interactors using RNAi and observed an increase in HIV infection, suggesting that those factors may play a role in inhibiting replication.

"Ultimately, our analysis of the host factors co-opted by different viruses using the same proteomic pipeline will allow for the identification of protein complexes routinely targeted by different pathogens, which may rep- resent better therapeutic targets for future studies."

[1] Jäger, S., Cimermancic, P., Gulbahce, N., Johnson, J., McGovern, K., Clarke, S., Shales, M., Mercenne, G., Pache, L., Li, K., Hernandez, H., Jang, G., Roth, S., Akiva, E., Marlett, J., Stephens, M., D’Orso, I., Fernandes, J., Fahey, M., Mahon, C., O’Donoghue, A., Todorovic, A., Morris, J., Maltby, D., Alber, T., Cagney, G., Bushman, F., Young, J., Chanda, S., Sundquist, W., Kortemme, T., Hernandez, R., Craik, C., Burlingame, A., Sali, A., Frankel, A., & Krogan, N. (2011). Global landscape of HIV–human protein complexes Nature DOI: 10.1038/nature10719

Photo: I'm crazy about soap bubbles this week. The macro lens can enlarge all the pretty color patterns and they are so, so beautiful. More bubble awesomeness to come later!

So mice can be vaccinated against HIV. What about humans, though?

I hope I can get away with yet another paper on gene therapy this week. You may actually have already heard about this one: it came out at the end of November and it had quite some resonance because the researchers claimed to have established lasting immunogenicity to HIV in mice‚ using, again gene therapy.

I have already discussed the potential use of gene therapy to cure HIV. In fact, the only human to ever be "cured" of HIV was a leukemia patient who, after receiving a genetically modified vector, developed HIV-resistant T-cells. In that case gene therapy was the only way to save the patient's life as he was dying of leukemia. It was quite an interesting study and I loved learning about it. However, in general, gene therapy is NOT a feasible way to end the AIDS pandemic. It's too expensive, too risky, and 2/3 of the infected people live in South Africa where even drugs are too expensive, you can imagine gene therapy.

No, the most efficient means to wipe out the virus, from both an economical and a clinical perspective, is a vaccine.

Okay, I'm biased. I work on HIV vaccine design. And when this paper appeared in Nature many colleagues rolled their eyes. "Too risky." "Too impractical." "It'll never work in humans." Which meant I had to read the paper. So I did.

From the abstract:

"As an alternative to immunization, vector-mediated gene transfer could be used to engineer secretion of the existing broadly neutralizing antibodies into the circulation. Here we describe a practical implementation of this approach, which we call vectored immunoprophylaxis (VIP), which in mice induces lifelong expression of these monoclonal antibodies at high concentrations from a single intramuscular injection. This is achieved using a specialized adeno-associated virus vector optimized for the production of full-length antibody from muscle tissue. We show that humanized mice receiving VIP appear to be fully protected from HIV infection, even when challenged intravenously with very high doses of replication-competent virus. Our results suggest that successful translation of this approach to humans may produce effective prophylaxis against HIV."

So, it is some kind of vaccine. And at the same time it's not. In a standard vaccine you inject a deactivated form of the virus in order to elicit antibody production in the host. With this new method, instead, you inject a virus which carries the genes for the antibodies. Instead of letting the immune system find a way to produce the antibodies, the researchers provided the "instructions" on how to make them: they injected into the muscle a viral vector containing the genes for the antibodies.

The vector used in the study is a self-complementary adeno-associated virus, which I discussed here. The researchers produced AAV vectors that either expressed luciferase (for the controls) or the neutralizing antibody b12 and administered them to mice through a single intramuscular injection. The mice were then populated with human peripheral mononuclear cells and then challenged with HIV. After the challenge, most mice expressing luciferase showed dramatic loss of CD4 cells (the cells infected by HIV) whereas mice expressing b12 antibody showed no CD4 cell depletion. Basically, the therapy was working.

They also tested a cocktail of historically known broadly neutralizing antibodies: b12, 2G12, 4E10, and 2F5. Again, after being adoptively populated with huPBMCs, the mice were

"challenged by intravenous injection with HIV and sampled weekly to quantify CD4 cell depletion over time. Animals expressing b12 were completely protected from infection, whereas those expressing 2G12, 4E10 and 2F5 were partly protected."

Finally, they repeated the experiment with one of the newest and most potent broadly neutralizing antibodies, VRC01, and found similar results, with higher protection established at higher doses of the vector.

The fact that the mice were challenged intravenously is quite impressive because mucosa routes present a bottleneck for the virus, whereas intravenous challenges are much more efficient in initiating the infection.

A number of things remain to be seen, the safety and efficacy of the therapy in particular. In this hemophilia B study the administration of intravenous AAV showed lasting results, even though the new genes were expressed at a low level. However, other scAAV studies have failed and, as an additional word of caution, we should not forget all the therapies successfully tested in mice that later failed in humans: assuming this technique passes the required safety checks, it still remains to be seen whether results in humans would be comparable to the mouse model.

Still. Despite my original bias, I confess I find these results pretty cool. Don't tell my boss, though!

Balazs, A., Chen, J., Hong, C., Rao, D., Yang, L., & Baltimore, D. (2011). Antibody-based protection against HIV infection by vectored immunoprophylaxis Nature DOI: 10.1038/nature10660

Photo: Sunset on Croc Rock and the Rio Grande. Shutter speed 1/25, focal length 38mm, f-stop 5.0, ISO speed 100.

Timing the AIDS pandemic and why it made history (Part II)

In Part I of this post I discussed the Science paper that proved HIV was the result of a cross-transmission from chimpanzees to humans. In that paper, Hahn et al. conclude with an open question:

"The timing of SIVcpz transmission to humans, leading ultimately to the HIV-1 pandemic, has been a challenging question. We know from analyses of stored samples that humans in west central Africa had been infected with HIV-1 group M viruses by 1959 and with group O viruses by 1963. But how much earlier were these viruses introduced into the human population? [...] It should be possible to estimate the timing of the onset of the pandemic by calculating the date of the last common ancestor of HIV-1 group M."

In a phylogenetic tree (see the definition I gave last time), the last common ancestor is the root of the tree: that's the "patriarch" of the sample if you will, the one sequence from which, one divergent event at the time, the whole sample originated. Phylogenetic analyses allow us not only to reconstruct the evolutionary history of the sequences, but also, if you have a rough idea of what the mutation rate is (i.e. how often new mutations arise) to time them. It's a technique often referred to as "molecular clock," which originated from the observation that the number of molecular differences between different lineages increases linearly with time and that substitutions accumulated according to a Poisson distribution.

Korber et al. used parallel computers to apply maximum-likelihood tree-building methods to the envelope sequences (the envelope is one of the HIV genes) from 159 individuals. They note:

"Although it is unrealistic to expect that HIV-1 evolution will always rigidly adhere to a molecular clock, it is, however, the average behavior of many sequences that we consider here, and our control estimates of known times were accurate."

To this they combined another data point: the year of sampling of the sequences used to reconstruct the tree.

(A) The phylogenetic tree used for the calculation. (B) The branch lengths from the tree plotted versus the year of sampling an dprojected backwards in time.

Once they reconstructed the phylogenetic tree, with the root sitting more or less in the middle, and thus at the same distance from the various HIV subgroups (the clusters marked with capital letters in panel A above), they plotted the branch lengths of the tree against time (panel B) and did a linear fit to extrapolate the time since the last common ancestor: 1931, with a 95% confidence interval of 1915 to 1941. Furthermore, testing a known HIV-1 group M isolate from 1959 gave an accurate estimate for the date of its origin, indicating that the assumptions of the method are reasonable.

Notice that 1931 marks the year the first HIV-1 lineage, the M-group, started to spread and diversify in humans. It does not tell us whether or not the virus was transmitted at the same time as it started to diversify. It could be possible that the virus cross-transmitted to humans earlier and remained isolated within a small population. Around the '30s socioeconomic changes would've allowed the spread of the virus:

"Strictly speaking, our estimate is neither an upper nor a lower bound on the date of the actual zoonosis. Rather, it is the approximate time of the bottleneck event that was the genesis of the M group and captures the moment of the beginning of the expansion of the M group. If the M group originated in humans, then this would date the founder virus of the pandemic."

Another important question is addressed in the following commentary by David Hillis:

"If HIV has been present in human populations since at least the 1930s (and probably much earlier), why did AIDS not become prevalent until the 1970s? The phylogenetic trees of HIV-1 indicate that the spread of the virus was initially quite slow‚ by 1950 there existed 10 or fewer HIV-1 M-group lineages that left descendants that have survived to the present. The epidemic exploded in the 1950s and 1960s, coincident with the end of colonial rule in Africa, several civil wars, the introduction of widespread vaccination programs (with the deliberate or inadvertent reuse of needles), the growth of large African cities, the sexual revolution, and increased travel by humans to and from Africa. Given the roughly 10-year period from infection to progression to AIDS, it was not until the 1970s that the symptoms of AIDS became prevalent in infected individuals in the United States and Europe."

B. Korber, M. Muldoon, J. Theiler, F. Gao, R. Gupta, A. Lapedes, B. H. Hahn, S. Wolinsky, and T. Bhattacharya. (2000). Timing the Ancestor of the HIV-1 Pandemic Strains Science, 288 (5472), 1789-1796 DOI: 10.1126/science.288.5472.1789