Page History

Note: The initial 7 minutes of John Mellors' talk has been omitted, due to an audio problem.

John Mellors: —Multicenter AIDS Cohort Study. Here are the PIs. Steve Wolinsky's picture is missing. It has nothing to do [00:00:30] with our recent disputes about ongoing replication. This was pulled directly from the MACS website. I did not edit Dr. Wolinsky's picture.

The MACS has made many, many, many important contributions. The major risk factor for incident HIV being anal receptive intercourse. (1) Incident infection is usually asymptomatic or mildly [00:01:00] asymptomatic. (2) They demonstrated that the natural history of HIV infection is profoundly heterogeneous, and I'll expand on that. (3) The very important finding that the risk of PCP (Pneumocystis pneumonia) or PJP in MSM is increased when the CD4 count falls below 200. (4) And beginning work on host genetic regulation of susceptibility and disease progression. (5)

So I had this foggy hypothesis: more pathogen, more [00:01:30] disease. Bob [Gallo], I didn't put the slide up here, but you and Flossie [Wong-Staal] published a paper saying that there wasn't much infection in the bloodstream by in situ analysis, that there were rare infected cells. (6) I was perplexed by this because as an ID physician, I knew that pneumococcal bacteremia in asplenic individuals was very high titer and rapidly lethal as it was for meningococcemia, [00:02:00] as it was for gram-negative sepsis, and in general, for tuberculosis. More tubercle bacilli, more advanced disease. I wondered, is this similar for HIV?

Here is our first data. We looked at plasma HIV and cell-associated HIV RNA. Here are five individuals who did not progress to AIDS and they had undetectable HIV RNA with a cutoff of 10,000 [00:02:30] copies per mL. Here are the individuals who progressed to AIDS, who had higher viral load and increasing viral load. Additional data were that 29% of this pilot study of 62 developed AIDS, and then a multivariate analysis of every known laboratory value having an RNA above 10,000 copies was the most powerful predictor of AIDS. We sent the paper off, and The Lancet [00:03:00] editor wrote back to me and said, "I can't imagine how this work would prove to be useful." You can't make this up.

[laughter]

I send it to the Annals [of Internal Medicine], and they accepted it right away. (7) I wanted to do a larger study. I asked the MACS leadership, and they said, "It's interesting, but it's a low priority and there are no resources." I looked locally for resources. [00:03:30] This is Holly and Afrouz Bazmi. Holly was in my laboratory, and this is her sister. I paid her sister out of my own resources to sneak up the back stairway and pull the samples from the MACS repository with permission and not in violation of any scientific principle. I paid her to come in after-hours and on weekends.

That resulted [00:04:00] in collaboration with Chiron, John Todd, and others. (8) The data from the Pittsburgh MACS showing this remarkable stratification of time to death by, in this instance, two baseline viral loads. What convinced me of the importance of viral load is in panel A and B, are two groups of individuals, one with CD4 above 500, one with CD4 below, [00:04:30] but the median CD4 is very similar, yet the survival is, obviously, quite different.

I was satisfied with that, but then suddenly, the leadership said, "We need to do the entire MACS cohort and test them for viral load, and look at prognosis." Enter Alvaro Muñoz, who is one of the most colorful and humorous individuals I've met in my [00:05:00] career, a delight to work with. He would say these crazy things like, "Maestro, call your mother, she will be proud." [laughter]

What was he talking about? He was talking about this. This is 1,600 individuals in the Multicenter AIDS Cohort Study. This is of a single viral load, and the stratification of survival over a 10-year period is truly remarkable. [00:05:30] Alvaro went on to make the famous skyline plots. (9) This is adding CD4 count at entry to baseline viral load either by bDNA or RT-PCR. This is the risk of AIDS at three years, six years, and nine years.

I wanted to finish up this story by clearing up some inconsistencies in the literature. [00:06:00] In 2007, a paper was published suggesting that viral load did not predict CD4 decline. (10) And so we reanalyzed all the MACS data, baseline viral load versus time to AIDS, and remarkably, one viral load measurement explains about 50% of the variance in outcome. (11) There's also the indication in the literature that CD8/38 RFI [00:06:30] is a better predictor of outcome. In this analysis, it was not a better predictor, and it's highly correlated with HIV RNA and the two combined at modest predictive value. Alvaro would call me and say, "Your viral load is the king. CD8/38 may be the princess." [laughter] And so the misconception from the paper in 2007 [00:07:00] by Rodríguez et al. was that a CD4 cell slope was an important prognostic indicator. In fact, CD4 cell slope, because it's such a variable measure, doesn't predict AIDS and only explains less than 3% of the variance of time to AIDS.

One other misconception in the literature is that there is a viral set point after infection. This study by [Robert H.] Lyles showed—ignore this panel, look at this panel—[00:07:30] that individuals who progressed AIDS in less than three years, three to seven, or greater than seven, or never developed AIDS after more than 10 years of follow-up, and the individuals who progressed rapidly, there's not a set point, the viral load continues to increase over time. (12) Also, in those who don't develop AIDS, the viral load declines. The two other groups are somewhere in the middle. Even the group within three to seven years has a positive RNA slope. [00:08:00] We should say with some caution that there's a set point, it has a positive slope, and rapid progressors, and a negative slope and non-progressors.

To validate HIV RNA as a clinical tool, there's two steps required. I told you about the first step as a prognostic biomarker. (8, 9) As important is to validate as a surrogate endpoint for treatment trials, meaning that a change in the marker is a surrogate for a clinical outcome [00:08:30] AIDS or death.

This was nicely shown by two studies. First, the VA Cooperative Study 298. (13) Is John Hamilton still here? I hope so, but I don't see him. And ACTG 175. (14) Basically, before viral load, we had to do huge clinical trials, 2,500 individuals, long-term followup, and terrible endpoints: 50% reduction in CD4, AIDS events, or death. [00:09:00] What Bill O'Brien and John Hamilton showed that a 75% decrease in HIV, a modest decrease, was associated with a 60% reduction in risk of progression to AIDS, highly significant, and held up in a multivariate analysis. David Katzenstein showed that a one log reduction in HIV RNA in ACTG 175 was associated with a 80% reduction in the univariate analysis as [00:09:30] a 65% to 70% reduction in AIDS or death in a multivariate analysis.

In conclusion, plasma RNA is arguably the best marker of prognosis and surrogate marker of therapeutic response that has ever been identified in medicine. Plasma HIV RNA quantification greatly accelerated the development, improvement, and [00:10:00] approval of ART. With that, I'll give you a smiling Jack-o'-lantern and remind you to get candy for the kids.

Let's move on to Viremia Chapter 2, and enter a John Coffin. John and I developed an acquaintance that went something like this, "You look like death warmed over (Geneva 1998)." That's because [00:10:30] I gave 11 talks in three days, and I won the Sputnik Satellite Award and vowed never to give a satellite talk again after that. But this is how we usually see John, saying, "That makes no sense," which is what he says to me and others frequently when we're meeting in the third floor of Building 535 in Frederick [National Laboratory for Cancer Research], which is the home of the HIV Drug Resistance [00:11:00] Program and now the Dynamics and Replication Program. On top of us are [Jeff] Lifson, [Jake] Estes, and [Brandon] Keele who look down upon what we do on the third floor. No, they're actually very, very nice. The DRP philosophy is very simple. It's just a natural progression of Cold Spring Harbor. There is not a nucleotide or amino acid in any retrovirus nor a step in a retroviral life cycle that isn't worth [00:11:30] arguing about for an hour, a day, or a lifetime. That's what we do. The other corollary to this, is much easier to interrupt somebody, to listen to what they have to say. [laughter] Here is the current group, led by Mary Kearney. John and I are consultants. They haven't kicked us out yet but that day will come. [00:12:00] 

Credit to John, you're going to hear from David [Ho] and Alan, you've heard a little bit. About 1995 the kinetics of HIV decay—which is essentially the plasma RNA, the decay or death of infected cells—was censored by the limit of detection of the available assays in about 50 copies per mL. (15, 16, 17, 18) John asked, "What happens below?" and I helped him and the team developed the "single copy assay," which was published [00:12:30] by Sarah Palmer et al. in 2003. (19)

We applied this assay to ask a variety of clinical questions. The one I was most interested in was the study Abbott 98-863 which was a comparison of lopinavir/ritonavir to nelfinavir. (20) There was a marked, highly statistically significant difference in the failure rate between the two arms. I asked, [00:13:00] "Does a difference in antiretroviral 'potency' impact persistent viremia on therapy?" The hypothesis was it would. We pulled out 130 patients of equally distributed between the two arms, who remained less than 50 copies following week 24, and asked, by the single copy assay, "Was there any difference in the degree of suppression?" In fact, there was absolutely no difference in the degree of suppression, and the failure rate [00:13:30] difference is likely due to the higher frequency of pre-existence of nelfinavir resistance mutants, or to the poor adherence to nelfinavir. We also looked at NNRTI (non-nucleoside reverse-transcriptase inhibitor) based regimens, and there was no difference. We concluded from this that current ART maximally suppressed viremia. We published that in PLOS Pathogens and showed that the pre-ART [00:14:00] viremia was the best predictor of on-ART viremia. (21) That the levels could persist for at least seven years and most recently in a large study showed that there's continued slow decay of residual plasma viremia, which caused me to go back and cross this out, stable viremia. (22, 23)

Here is the sum of the work. We have a Phase III decay with a half-life of 2/3 of a [00:14:30] year. A Phase IV decay-- We initially thought it was a zero slope but there is a slope with a half-life of about 11 years. About 70%, 80% of persons on effective ART have detectable viremia. It correlated, as I said with pre-ART viremia, that's the only correlate we've been able to find, median of one copy per mL, which makes it difficult to detect and quantify, slow decay over with a half-life of over 10 [00:15:00] years.

We also, in collaboration with the ACTG, looked at whether adding an antiretroviral drug raltegravir affected persistent viremia. In an immediate versus deferred intensification trial ACTG 5244 published by Raj Gandhi et al, there was really no effect of raltegravir intensification. (24) There were some issues with the original version of the assay and we refined it [00:15:30] such that it targets the most highly conserved region of the proviral genome and has greater sensitivity and fewer false negatives. (25)

Last chapter, origin of persistent viremia. As you well know, by Maldarelli et al., and Wagner et al. in Sciencein 2014, the identification of expanded infected cells or clonally expanded infected cells [00:16:00] was found in one individual patient one, the individual had persistent viremia that matched the proviral DNA. The persistent viremia was identical sequence, there was also a drug-resistant population that went away after the regimen was changed to cover this, and the individual had persistent viremia that looked clonal. (26, 27) Bob Siliciano and many others had described what was called a predominant plasma clone. [00:16:30] We saw that in this individual. Integration Site Analysis was performed. 3% of the expanded clones in this individual had a ambiguous site in the human genome. One of three sites. We couldn't map it precisely to one of those three sites but a specific location, but that was redundant in three locations. Amplified [00:17:00] the full-length provirus and much to our surprise between the integration sites, the provirus appeared to be intact. Amplified it in two halves, transfected it into permissive cells, and then passed it into CD4-positive blast. It grew vigorously, it also grew in TZM-bl cells and was inhibited by efavirenz. We went back to the patient, got CD4 cells isolated [00:17:30] virus, sequenced it, and the sequences of the isolated infectious virus matched the plasma viremia and the proviral DNA. (28) This was the first example of clonal expansion of an infected cell producing infectious viremia.

You might ask, are there other individuals who have expanded infected cells that have intact proviruses? The answer is yes. Here's just one other example. [00:18:00] A plasma RNA by single genome sequencing, and the colored diamonds are viral outgrowth wells. Notice that they match identically and also match DNA sequences. Multiple identical drug-sensitive sequences in plasma HIV DNA and virus outgrowth wells. In this individual 16% of all the DNA, we [00:18:30] found, matched the plasma sequences. Here is some other expressed but likely defective proviruses because they didn't grow or were dominated in the viral outgrowth assay. In eight of nine donors studied with persistent viremia on ART, we found clonal viremia. The plasma viral sequences match infectious virus recovered from virus outgrowth in four of the eight. We have a persistent clone that produces infectious [00:19:00] viremia and its spread is blocked by ART.

What are the implications? The clones harboring intact, expressed or inducible proviruses are part of the reservoir that needs to be targeted to cure HIV. What proportion of proviruses are expressed isn't known but Drew Musick has a poster here showing that about 3% to 5% of proviruses in these clones are expressed. We need to know the dynamics [00:19:30] of infected cell clones, the proliferation rate, and importantly how they escape in the immune host. We are just beginning to start to look at the impact of curative interventions on clonal-amplified proviruses, especially intact ones. I talked with Bob Gallo about this and it's possible that any of these interventions could actually expand the clones.

Finally, there's an ongoing [00:20:00] controversy about a continued replication during ART in lymph nodes. Suffice it to say that this is a battle that will be played out over the ensuing months, and probably hopefully come to finality at CROI (Conference on Retroviruses and Opportunistic Infections). We've downloaded the sequences from this paper and once we collapse all the identical sequences, we don't see any evidence of evolution over time in lymph nodes, plasma or PBMC.

In this [00:20:30] individual, you might suggest that there is some evidence of [unintelligible 00:20:35], but this is the individual who wasn't suppressed. I'll close by thanking the more than 23 colleagues, the volunteers, and the funding agencies. Thank you very much.

[applause]

Alan Perelson (Moderator): Well, thank you, John. This talk is now open for questions.

John: Veffa.

Veffa Franchini: Yes, does any of you know which cells carry this thing [00:21:00] [inaudible 00:21:00]?

Alan: Repeat the question, please. Why don't you repeat the question?

John: Veffa has asked, do we know which type of cells these are, and we don't. We're working hard on that. I suspect there may be some skewing towards a specific population, but I think any cell population could have this. I'm most concerned that a stem-like memory cell has integrated provirus that could expand indefinitely. I think it's going to be highly variable. Maybe the effector [00:21:30] because they're most expanded effector memory, but any of the others could.

David Gludish: Just related to that, you probably thought about what would cause the cells to be able to escape clearance by apoptosis, but you do have the sequence to the virus, any clues there about whether BPR is defective or what would allow that—

John: Seems to be intact. No obvious defects, [unintelligible].

Participant 2: [inaudible 00:21:55]

John: We're really just [00:22:00] starting those. There are multiple mechanisms, including CTL escape, but one possibility is relatively apoptotic resistance cells.

Alan: One more question from Bruce.

Bruce Walker: John, how accurate a picture do you think we're getting from the peripheral blood, and do you think we really need to press towards really examining lymphoid tissues?

John: That question is being examined with vigor, what I [00:22:30] say is very preliminary, but we don't see any difference between lymph nodes and blood in integration sites, expression, or the sequences that are being expressed. That's contrary to a lot of scientific cognoscenti's feeling, but this is a critical question. So far it looks like we can tell a lot from the blood.

Alan: Well, thank you very much, John.

[00:22:54] [END OF AUDIO]

Citations

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Index

• 1.5 John Coffin — The Origin of Molecular Retrovirology
• 12th International AIDS Conference, Geneva, 1998
• 2.4 Robert Gallo — Discoveries of Human Retrovirus, Their Linkage to Disease as Causative Agents & Preparation for the Future
• 4.0.1 Jeffrey Lifson — Session 4, Introduction 1
• 5.1 Flossie Wong-Staal — Discovery of Human Retroviral Transactivators
• 6.3 Bruce Walker — Role of T Cells in Controlling HIV Infection
• 8.2 David Ho — Unraveling of HIV Dynamics In Vivo
• 8.4 Robert Siliciano — The Challenge of the HIV Reservoir
• Abbott Laboratories
• adherence, patient compliance
• AIDS Clinical Trials Group (ACTG)
• analogy
• Annals of Internal Medicine (journal)
• antiretroviral therapy (ART)
• Bazmi, Holly
• blood — banks, donors, plasma, screening, transfusions, clotting factors (factor VIII), PBMCs 
• Chiron (1981–2006)
• Cold Spring Harbor Laboratory (CSHL)
• Conference on Retroviruses and Opportunistic Infections (CROI)
• EFV (efavirenz)
• Estes, Jacob D.
• Frederick National Laboratory for Cancer Research (FNLCR, NCI-Frederick)
• funding and grants
• Gludish, David W.
• Hamilton, John D.
• hypothesis
• infectious disease (medical specialty)
• Katzenstein, David A.
• Kearney, Mary F.
• Keele, Brandon F.
• Lancet (journal)
• lopinavir
• Lyles, Robert H.
• lymphatic system (lymph, lymph nodes, etc.)
• MSM (men who have sex with men)
• Multicenter AIDS Cohort Study (MACS, 1984–2019)
• Muñoz, Alvaro
• nelfinavir (NFV)
• non-nucleoside reverse-transcriptase inhibitors (NNRTIs)
• O'Brien, William A.
• Palmer, Sarah
• peer review
• Pittsburgh, Pennsylvania
• PLOS (journals)
• Pneumocystis pneumonia (PCP)
• provirus
• raltegravir (RAL, Isentress)
• RTV (ritonavir, Norvir)
• scientific competition and collaboration
• scientific controversy and consensus
• sensitivity and specificity; false positive, false negative; biological specificity
• sequencing
• Session 7: Prospects for an HIV Vaccine
• Todd, John A.
• transfection, transduction, viral vector
• viral load
• viral reservoir, viral latency, disease reservoir
• Wolinsky, Steven