|Not So Unforgiving After All?
Viral Load Post TTI Mega-HAART
in WT-Shifters & Non-Shifters
|N||26/39 (66.7%)||13/39 (33.3%)|
|change in RNA (Wk8, mean)||- 2.9||- 0.78|
|RNA > 500 (Wk24)||19/24 (79%)||1/9 (11%)|
total treatment interruption; WT
(see related report)
In HIV infection, the role of the thymus remains both hotly disputed and poorly understood -- whether its role in leading to the clinical manifestations of disease or in rebuilding an already HIV-ravaged immune system. Along with a handful of other pioneers in this work, Joseph "Mike" McCune has been at the forefront of immunological research for over a decade.TAGline checked in with him this summer for an update of his most recent research and for a hint of where we might be headed -- or need to go.
It seems like we know more now about the thymus in HIV infection than we know about the bone marrow. Am I wrong?
I would say that they are both understudied. It has been known since the mid-1980s that the bone marrow is infected by HIV. So even before we had, say, AZT trashing red cells many people came in with bone marrow abnormalities. This has been well documented -- but not very vigorously pursued. It's a difficult thing to do.
For obvious reasons, I guess. But if the source of T cells is actually the bone marrow -- with the thymus serving only as the "boot camp" or "finishing school" for the progenitor cells which emerge from the bone marrow's stem cells -- would correcting the pathology in the thymus not matter as much if the migrating cells from the bone marrow are already not fully functional?
That's right. If, for example, you had a fully functional thymus but no bone marrow, it wouldn't help. It'd be like having a car without gas.
Let's talk briefly about the work you presented at the Lago Maggiore resistance workshop last summer and which was published last June in JCI [Journal of Clinical Investigation]. Basically you showed what you called "abundant thymic mass" in about 50% of the HIV-infected adults you looked at. Many over age 40 had it, and in people younger than 40 with CD4 T cell counts between 300-500, a whopping 93% of them showed evidence of thymic mass (on CT scans). Did anything about that research surprise you?
Oh yeh! The reason that we knew more about the bone marrow than the thymus until that study is that, up until that time, [it was widely assumed that] the thymus would not be present at all in adults.
Yeh, that was the prevailing ... Mark Harrington called it the "hoariest dogma" of immunology.
We had actually studied, using these chimeric mice, in the early 1990s ... we spent a lot of energy studying ... how HIV can destroy the thymus. And it does so because it infects CD4 positive cells, which are key cells. So we had those data, and there were data in SIV-infected monkeys that the thymus is destroyed, and there were data in HIV-infected babies that the thymus is destroyed. So when it came to adults, the "hoariest dogma" said that the thymus would be much smaller -- if it was there at all. And if HIV was around, then it was most certainly not going to be there because HIV destroys the thymus, right?
Our study was set up, actually, to understand whether or not the hoariest dogma was true -- because it had not yet been studied in HIV-infected adults. And what that paper showed, basically, was that by relatively crude measures (CT scanning and analysis of these so-called "naive T cells" by phenotyping in the peripheral blood) that there does appear to be thymic mass and function in HIV-infected adults. That was surprise number one. And surprise number two was that it's even easier to find evidence of thymic tissue and function in HIV-infected compared to age-matched HIV-uninfected adults.
Yeh, I didn't catch that in your Lago Maggiore presentation, but when I read your JCI paper last night, that's really amazing.
Yeh, we were surprised by that. Of course there is still a fair degree of uncertainty about whether what we're looking at is truly functional thymus. (CT scanning is not capable of distinguishing between thymic tissue and tissue that might simply be a conglomeration of inflammatory or malignant cells.) We know now, however, that it is possible to look at other surrogates of thymic function, such as TREC (deletion circles) as have been studied by Danny Douek in Rick Koup's lab, by Sharon Lewin in David Ho's lab, and by Jean-François Poulin working with Rafick Sekaly and myself. Study of these surrogates also suggests that thymic function is present in adults, even some of those who are HIV-infected.
So they sort of corroborate each other.
Yes. I think the datasets that are coming back now all seem to be consistent with one another. Which is to say that the thymus is functional in many adults; that the function decreases as people get older; and, most importantly, that there is a tremendous amount of heterogeneity amongst HIV-infected adults: some have abundant thymus and others have little apparent thymic function. So, when considering the effects of HIV and treatment in adults, an important variable might be whether the individual has thymic function or not.
And there's really no way to predict this based on age, viral load, CD4 cell count or duration of HIV infection. Is that right?
Partly. We did notice that more abundant thymic tissue was present in young, HIV-infected adults and, amongst those, especially in individuals with falling CD4+ T cell counts, between 300-500. As you mentioned, over 93% of such individuals having abundant thymic tissue, which is extraordinarily high.
So do you think the thymus "realizes" that something is going on and so it revs itself up and tries to compensate for the destruction or disappearance of these T cells -- and then at some point either HIV gets the best of it or it just can't keep up?
Good, yeh. So the working hypothesis is, as you say, that there is compensatory positive feedback on the thymus -- and maybe also on the bone marrow when T cells are depleted in the periphery. And as long as the thymus can compensate (by making more cells), then the immune system in the periphery will not collapse -- because you'll just make more cells. Now ... we may as well talk about it now, because this is where the "tap" and "drain" debate comes in. The tap is now the thymus: it's producing more cells. And when the "drain" is open, more cells are made in order to compensate for the fact that the "sink" is emptying faster, okay?
Now David Ho, when he first postulated tap/drain, talked about exhaustion. The tap became exhausted. Right? And could no longer compensate, if you will. But "exhaustion" needs to be defined biologically; cells don't "get tired." If they do, there's something called senescence... after a certain number of divisions...
Yeh, that was the telomere paper.
Telomeres, right. Yes, it's possible that they might get so old -- that they've divided so many times -- that they can divide no more. That's one physical correlate of exhaustion. Another correlate, which I think is supported more squarely by the data, is death. You know, "What would die?" Well, the tap would die. We know, as I mentioned, that HIV infects the thymus and kills the cells that are in it. Now, if the thymus is uninfected, it can work. Yet eventually, it would appear to us, that HIV is going to get sort of homogenized throughout the body. And it stands to reason even though there's a thymic-blood barrier in much the same way that there's a blood-brain barrier that the thymus is going to get infected by HIV. And once the thymus is infected by HIV, all bets are off. This compensatory increased production is not going to happen. And therefore the system is going to have destruction in the periphery and no production coming out to sustain the loss of cell.
This in fact is why in the Nature Medicine paper on production we state, my colleague Marc Hellerstein and I, that the disease is more a problem of production. That is to say, you can have the virus, but as long as the production systems are working there is no problem. But as soon as the production systems are hit, then you have a disease -- because the T cells go away and are no longer replaced.
And you're not alone. Fauci, Pantaleo, Miedema all seem to be advocating for an increased emphasis on the systems of production -- or "tap."
We're all still learning. We talk a lot about this. My scientific background has led me to work on the heterogeneous populations of blood cells that are found in the body. To me, it's part of my background to think about different types of cells and how they might be affected differently by HIV. Both "sides" in the debate are right -- some of us are simply pointing out that it's important to understand HIV's effects on progenitor cells as well as to think about the destruction of more mature cells.
So HIV's attacking the immune system from both ends?
Right. Now, there are several instances in which that doesn't happen with a lentiretrovirus, and these are very cool. One is in SIV. SIV infects a rhesus macaque, and causes basically AIDS. The T cells in the periphery go away, the animal gets sick and dies. The same virus, SIV, infects sooty mangabeys (another nonhuman primate) and what happens is NOTHING! The virus can go into the CD4 cells and kill the CD4 cells in the sooty mangabey, but the CD4 count goes down only slowly -- if at all. And one possibility, which is being pursued by a number of labs now, is that in this instance the virus has figured out how to kill mature cells but not how to kill the progenitor cells. And in this setting, actually, sooty mangabeys serve as a host for SIV in which the virus lives happily. So it's not a pathogen-as it is in the rhesus macaque -- it's a sort of a part of the ecosystem, instead.
So does that mean that the stem cells of these animals are naturally resistant to SIV -- and that we must wait for the day when gene therapy research has succeeded in its attempts to develop HIV-resistant progenitor cells?
Well, maybe it's that they're resistant but another possibility is that there are different types of HIV or, in this case, SIV. And that some are tropic more for progenitor cells than for mature cells and vice versa. So yeh, either possibility is right. A few, like Mark Feinberg, are working on this -- and Bob Grant.
But the other thing that's arisen, which I'm sure you're aware of is in HIV-infected adults who are "failing" therapy with high viral loads but who have viruses that are associated with rising CD4 counts. Now how the hell does that happen? This looks exactly like the SIV-infected mangabey to me! You have a virus which is present but it doesn't appear to be causing depletion of T cells the way the virus used to do. Cheryl Stoddart in my group has been studying viruses from such individuals with François Clavel. In Chicago in February they reported these viruses don't infect and kill the thymus.
So our working hypothesis is that in these individuals who have this virus/CD4 "disconnect," we're dealing with viruses that can kill CD4 cells in the periphery but which don't affect the systems of production (like, for instance, the thymus) as efficiently as they used to do. So the system can continue to make cells and pour them into the periphery even though there are lots of cells being destroyed there. Here, in other words, the "tap" is not getting tired.
Then, the hypothesis is that, what, there's something about one of these classes of drugs or the use of several classes in combination that restricts HIV to gnawing away only at the peripheral end of the T cells and prevents it from being able to affect production as much?
Hypothesis. That's the hypothesis behind the paper presented by Dr. Stoddart in Chicago. What was done there was based on work with a molecularly-cloned virus called NL43 (a CXCR4-utilizing synctia-inducing virus which we've used a lot). Put into these SCID-hu mice, this virus replicates quickly and destroys the thymus. François Clavel replaced the gag region of NL43 with gag that came from mutated regions of the protease gene of patients who had been treated and who had become resistant to either ritonavir or saquinavir. So we now had two viruses: one that was wild type NL43; the other was recombinant virus that included gag and protease regions from these patients that were resistant. The first virus went in and killed the thymus. The second virus went in and replicated slowly but didn't cause any cytopathicity in the thymus.
The only difference was the insertion of these regions.
So we can make protease resistant virus work for us?
Well, maybe. I mean, who knows?
Is there any degree of confidence that these beneficial mutations would remain stable, though, over time?
No, we don't . We have no ... I mean this is an area which is being newly investigated.
Very exciting, though. I missed that in Chicago.
I think a lot of people were surprised by this finding when Cheryl gave the talk. We certainly were, too. And we're now very eagerly doing more studies on it. In general, I think the idea would be that virus from these HIV-infected individuals may have "evolved" to approximate the situation of SIV in the sooty mangabey -- that of a pathogen which can co-exist relatively well within the host.
In nature, most microbial agents live in harmony with their host. In the situation of SIV with sooty mangabeys, you have a case of a lentiretrovirus that can be a pathogen which lives relatively well with that host. It's an important question to understand why and if that can happen with HIV. We're trying to approach it from the angle of differential pathogenicity to the systems of production.
I need to bring this around to treatment research agenda and therapeutic applications at some point. And I was wondering, I guess there were two papers in Geneva on thymus transplants and I actually, I had remembered, maybe there were two or three, that they all pretty much were failures but then I saw in Brenda's report for HIV-Plus that the people who were more immune compromised did not reject the transplants.
Yes, I know those data are coming out.
I just wonder that if maybe with all the data that your group and Koup and Sharon Lewis at Aaron Diamond ... is research into thymic transplant less urgent now? Or is it possible that it won't even be necessary if we have these demonstrations of the thymus being active?
The way I look at it is this. Let's say we have an assay for thymic function that everyone agrees is good -- we don't have that yet, but let's say we do. I would imagine we'd apply that assay to HIV-infected people when they present to ask the question, "Do they or don't they have a thymus?" And we'll have two groups: one will have demonstrable thymic function and one won't. Okay? And of those that do, some of them will have, you know, rip-roaring function and the others will be left with only a trickle of production.
Now, I'd guess that those who don't have thymic function will need to be treated differently than those who do. For instance, individuals in both groups may end up with an increased T cell count after therapy, but if the T cells do not arise from the thymus, they may not have a diverse T cell receptor repertoire. If so, these patients may still be prone to the problems associated with immunodeficiency. In other words, an increased CD4 count after HAART without a diverse T cell receptor repertoire may not be much help. So, in this scenario, we need to think about why they don't have a thymus. We've already noticed people who have increased their T cell counts post-therapy
No, just their memory cells, actually. They get therapy. Their CD4 count goes up, and they end up having or getting still things like CMV retinitis. So an increased CD4 count after HAART without a diverse T cell receptor repertoire may not be much help.
But that's not what we're seeing clinically -- for the most part. Pretty much across the board we're seeing surprisingly competent immune reconstitution -- even if these new T cells are not emerging from the thymus. How do we square this "no diverse repertoire" theory with the current clinical reality?
In general, we are indeed seeing a decreased incidence of opportunistic infections in patients who are effectively suppressed on HAART. Immunologic studies (e.g., those done by Krishna Komanduri in my lab and reported last year in Nature Medicine) indicate that such reconstitution is associated with increased T cell responses against these pathogens (e.g., CMV). Presumably, these T cells are present before HAART was initiated (i.e., they didn't come recently from the thymus) and are able to respond to the pathogens once HIV is suppressed. But this is by no means the rule, however: Krishna, working with Judith Feinberg (U Cincinnati) and Jay Lalezari (San Francisco), has noted that some HAART-treated patients have CD4 T cell increases and yet continue to have CMV retinitis; these patients, of note, lack anti-CMV T cell responses.
We worry that these patients may be the ones who lack a diverse TCR repertoire (and possibly a thymus) and that they represent the tip of a larger iceberg. More ominously, we are concerned about the implications of these observations in the context of long-term survival post-HAART: those individuals without a diverse TCR repertoire may be unable to effectively combat new antigenic exposures in the future, e.g., from other infectious agents, malignant cells, and the like.
So we should expect a new gay malignancy epidemic down the road. Great. But why might some individuals still have functioning thymic tissue and others not-and how could we address this therapeutcially?
I see two possibilities. One is that these individuals lack the feedback mechanisms that turn the thymus on. Another is that they lack the thymus upon which that the feedback mechanisms might operate. So let's take those in order: If they lack the feedback mechanisms, then provision of the feedback mechanisms might be sufficient.
Well, we don't know yet. That's what we're trying to figure out. But by analogy, erythropoetin causes more red cells to be made in the bone marrow okay? It's made in the kidney. People that have bad kidney disease have anemia. You know why? Because they don't make EPO.
And the way you treat that is not to give them a bone marrow transplant. The way to treat that is to give them EPO.
So we have to find the "EPO" for the thymus?
There you go. First, though, we have to understand whether feedback on T cell production in the thymus can occur, as it does in the example of EPO. If there is such feedback, then we have to understand how it operates. That, in my mind, is an extremely important area of research -- precisely because of the potential therapeutic implications.
So we don't even know if there is, in fact, feedback?
No. In fact, everybody thinks there's not. First there's not a thymus, okay, that's one dogma. The other dogma is that the thymus is a little homunculus that does what it does on its own and then dies. So that's the second hoariest dogma... Since 1960 or so... There have been a lot of experiments -- most of them in rodents -- done on this. I mean lots -- to ask the question, "Is there feedback?" and the answer is "No," that "there's not." But I'm not convinced yet that this is right in the setting of HIV-infected adults.
So then, to continue, if we find out there's not a feedback problem, there are going to be some people who lack thymus. Now, in the thymus there are several things that they might lack. One thing they might lack is the progenitor cell that makes the thymocytes. In such people, for instance, the bone marrow might be trashed, and the thymus doesn't have any of these cells upon which feedback could operate. In these people maybe a bone marrow transplant would help. Because you could give them new stem cells that would then give rise to new progenitors that would go to the thymus, and feedback could work on those progenitors and you would make more T cells for the periphery.
Finally, of those who don't have a thymus there could be some who lack what's called "thymic epithelial tissue," the collection of cells which define the structure of the organ. And that, actually, is what's being transplanted now. For such people, maybe we could figure out how to do transplant of thymic epithelia tissue in a way that works-in much the same way that we provide transplants of kidney or liver. Alright? I guess my basic point is that I don't think all HIV-infected adults with poor thymic function will have the same underlying lesion. Once we have better definitions for thymic function, we'll find that patients will fall into subgroups and that the subgroups will require different therapies.
One quick question: extrathymic maturation of T cells.
Mario Roederer [Stanford University] has concerns that new naive T cells that mature outside the thymus do not have the diversity and function of new naive cells that come from the actual thymus. Is that your feeling?
That is the prevailing wisdom. Probably right. The situation for instance with di George syndrome (where there is no thymus) indicates that cells made extrathymically are not diverse. There are elegant studies done in frogs and birds by Max Cooper which indicate that if you remove the thymus early in life (like in utero), T cells are not made later. That is, there is no other source of T cell maturation. It's possible, though, that extrathymic T cell maturation could occur in settings of unusual stress. A number of labs (including ours) are asking that question. You know, biology is incredible in its ways of coping, right?
In the hematopoetic system, for instance, the bone marrow is normally the place in you and me -- adults, that is -- where these multilineage stem cells reside, okay? But if the bone marrow gets trashed-by radiation or chemicals or disease -- other organs that usually do not operate to be hematopoetic organs can turn on as hematopoetic organs -- including the liver, the spleen, even the thymus. This is called extra medullary hematopoesis. ("Medullary" stands for the bone marrow.) So, in settings of stress, EMH clearly occurs.
So what a number of us are asking is if, in situations of stress on the T cell system, if extrathymic T-poesis might occur. Most of the experimental models that have been analyzed to date are not stressed. Okay? So the patient in di George is cuddled. Little baby, right? Kept out of the way of harm. The frog and the bird that were studied in utero? Not stressed. So there remains the possibility that in certain pathologic situations extrathymic maturation might occur in a way that provides a diverse repertoire. But, to date, it's not been clearly shown.
May I ask one last question? People are talking about the potential for hGH, bringing back thymosin alpha, thymopentin. Do you think any of these might have a role?
Sure. Here's why. It's possible that these represent part of, or a adjunct to, whatever this feedback loop on the thymus are.
And one of these might be the thymic "EPO" we're looking for?
Maybe. We don't know. There's certainly suggestive data that cortisol, for instance, is a steroid hormone which is bad for the thymus. It causes the thymus to involute. Testosterone does the same thing.
In the mouse interferon-alpha appears to be a potent negative regulator of thymopoesis.
And there are, in the literature, indications that other factors might actually promote thymopoesis. Growth hormone is one of those. IGF-1 (insulin growth factor), a mediator of growth hormone, is another.
These are in vitro data?
Most work has been done in vitro, but there are some anecdotal data in people as well. Patients who have acromegaly, for instance -- who have too much growth hormone -- can have big thymuses. It's all sort of still at the level of anecdote. And there are not very many -- if any, in fact -- good studies in humans because, you know, remember [he says wryly] "the thymus is not present in adults"; even if it's present, "it's not regulated." Right? So as a consequence of these prevailing "wisdoms," there have not been many studies to evaluate these questions about thymic hormones or other hormones which might act on the thymus. But there's more and more interest in this area now, and I think it's well worth the time to evaluate it. Because it would be attractive to be able to use commercially available products to promote thymopoesis.
And if people become more focused on the tap than they are currently on the drain, are the therapeutic implications significantly different?
Yes. Because if you're focused only the drain, you're mainly trying to figure out how to make a plug.
Which is ... antiretroviral therapy?
The plug in the case of the drain is antiviral therapy, right? If you're also thinking about the tap, then you might think of ways of making the tap work better, right?
Which would get us back to the thymic factors and the "EPO" for the thymus?
Or ... which promote production better. There might also be particular types of viruses that are especially cytopathic towards the progenitor cells. And there might be some drugs that work better against those viruses than against others. This is highly speculative, for HIV, but it's not at all for most other viruses. There is no such thing as another virus that has only one type, okay? There are some types of influenza viruses that are associated with clinically severe disease and others that are not.
Human papilloma virus (HPV), right? The virus that causes warts? In 1915 there were eighty different subtypes of HPV identified; some caused just warts and some caused cervical cancer. So it seems to me to be not outside the realm of possibility that there might be different subtypes of HIV -- not just X4 vs. R5 or SI vs. NSI -- but subtypes that have tropisms that are particular for stem cells or other progenitor cells. If so, there might be selective treatments that could be aimed at them. In other words, by focusing on the systems of production, we might also arrive at different ways of thinking about selective virus killing -- and ways to stop it.
Thanks Mike. A pleasure as always.
Interest in the virologic and immunologic effects of antiretroviral treatment interruption has been growing ever since Franco Lori first presented data on the by-now notorious "Berlin patient." And a fascinating presentation, by Dr. Veronica Miller of Frankfurt, Germany, at the Salvage Therapy meeting in Toronto earlier this year stirred things up further still: viral genotype reverting to wild-type during a median two month unplanned treatment interruption in 26/39 (66%) HIV-infected individuals treated at a Frankfurt HIV clinic.
Martin Delaney, Linda Grinberg and Mark Harrington held several recent discussions about the concept of a possible collaboration among FAIR (Foundation for AIDS and Immunologic Research), Project Inform and TAG focusing on a protocol, protocols, or workshop on the topic of research on structured drug holidays in HIV infection. Pursuant to these discussions, at the Forum for Collaborative HIV Research/NIAID/Project Inform/TAG co-sponsored workshop on The Challenges of Clinical Trial Design in Evaluating HIV Antiretroviral Use in Heavily Pre-Treated Individuals, Linda, Mark and Ben Cheng met with Veronica Miller and Schlomo Staszewski of the Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main in order to further explore the idea of a workshop or protocol on "drug holiday" research. Mark Harrington reports.
Veronica reviewed her research to date and defined some outstanding questions. The "drug holiday" research was not planned; the clinicians simply decided empirically to offer some patients a total treatment interruption because they had run out of options and were sick of taking scores of toxic pills every day to no effect. There are several other approaches, such as looking at a "drug holiday" when the patient's virus is well controlled as opposed to a rescue therapy situation. In addition, there has been interest in so-called "pulsed-dose therapy" since last year's TAG/amFAR symposium on HIV reservoirs and Franco Lori's presentation at Chicago in 1999. In Toronto, we decided to clarify terminology. "Drug holiday" is an easily abused concept, as shown by recent Glaxo Wellcome advertisements for Combivir, conflating "drug holidays" with non-adherence. Therefore, at lunch, we decided to take a stab at inventing some clearer definitions.
In Toronto, it became clear that there is a good deal of as-yet-uncoordinated structured drug holiday research already underway or in the late planning stages. So far, however, there has been no effort to coordinate the work, to ensure that the protocols generate useful comparative information, or to assure that they do not duplicate approaches, waste resources, or leave critical gaps which could be addressed up front. For example:
Many more studies are in the planning stages:
We discussed whether it might be possible to incorporate these issues into Project Inform's Eighth Immune Restoration Think Tank (IRTT-8), planned -- according to Ben -- for Chicago in October 1999. Generally it was felt that this was too far away, given the accelerating pace of the research and the patient interest. A separate, short and focused workshop, preferably held sometime in the summer, might be more to the point. We felt that, given the urgency of the issue and the difficulty of scheduling a long meeting soon, a meeting of two-and-one-half days might be ideal in length
After a conference call and some preliminary e-mails, we decided to schedule the workshop for late summer somewhere in the Boston/Cambridge area. Both Martin Markowitz and Veronica Miller agreed to serve on the steering committee, and Project Inform secured a West Coast researcher to do so as well. A summary of the meeting is scheduled to appear in next month's TAGline.
In Toronto, Dr. Veronica Miller presented an expanded dataset derived from results she had shown in Glasgow (November 1998) and Chicago (February 1999) on the Frankfurt HIV clinic cohort. The initial study impetus was not a drug holiday per se, but rather the introduction of so-called mega-HAART in heavily pre-treated, highly drug-resistant HIV patients after a variety of clinical courses, some of which had included a total treatment interruption (TTI) due to the lack of available viable treatment regimens. In Glasgow and Chicago she showed that three out of five patients (60%) who chose to undertake a TTI before initiating mega-HAART experienced a reversion of viral genotype to wild-type. At the Second International Workshop on Salvage Therapy for HIV Infection, Veronica Miller expanded the dataset to demonstrate that the same result occurred in 26/39 (66.67%) such patients. Moreover, among those whose virus shifted to wild-type, the introduction of mega-HAART caused a significant and profound drop in viral load and was not always associated, at least in the short term, with the re-emergence of genotypic resistance or drug failure.
Traditional retrovirologic dogma had held that the virus is usually, if not always, "genetically unforgiving", that the emergence of drug-resistance is a one-way street, and that even if wild-type (WT) virus emerged during a drug holiday, resistance was sure to re-emerge rapidly once the selective pressure of any given drug or a cross-resistant drug analogue were reimposed. Miller's data suggested that, at least in the short term, in a surprising two-thirds of patients who'd experienced a TTI, this was not the case. Her presentation, undoubtedly the most provocative and intriguing one at the Salvage Therapy Conference, generated a considerable stir.
Veronica presented data on 85 highly pretreated patients who were given mega-HAART. Of these, 81% had previously received six or more antiretrovirals. The baseline viral load, at the time they initiated mega-HAART (often after a TTI) was 5.21 log10 and the baseline CD4 count 108/mm3. The mega-HAART included six (64%), seven (30%) or eight (6%) drugs. Median follow-up was twelve months. Of the 85 people enrolled at Frankfurt, ten had experienced a total treatment interruption (TTI) of at least two months prior to initiating mega-HAART. Miller had pre- and post-TTI viral isolates from nine of these ten patients, and sent them off to Virco for resistance testing. Of these nine, six (66.67%) had experienced a shift to wild-type (WT) virus measured both genotypically and phenotypically. Their isolates had previously demonstrated 100-fold resistance to AZT and 3TC, 1,000-fold resistance to the NNRTIs and high-level resistance to the protease inhibitors as well.
Because of these intriguing results, and to increase the relatively small dataset, Miller set out to do a drug holiday analysis on fifty patients from the Frankfurt cohort. She looked retrospectively at all patients treated since January 1996 who had received at least two protease inhibitor-containing regimens for over one year and who had discontinued treatment for at least two months. Of fifty such patients, pre- and post-TTI resistance samples were available for 39 patients. By phenotypic and genotypic analysis, 26/39 (66.67%) of these TTI-to-mega-HAART patients experienced a complete reversion to wild-type (WT) virus during the TTI. Their virus had previously been resistant to a median of eight drugs (range 2-11). There was no shift in 13/39 (33.3%), who had received the same median number of previous antiretrovirals.
Among the 26 WT-shifters, the change in CD4 levels was not dependent on baseline viral load. Both groups lost about 125 CD4 cells. Among non WT-shifters, viral load increased more dramatically in those beginning with a low viral load. (Viral load appeared to rise to an equal physiologically possible level in both WT-shifters and non-shifters.)
Among the non-shifters, the CD4 loss was small if baseline viral load was low (-24 cells/mm3) and large if baseline viral load was high (-88 cells) -- the expected result. However, according to Veronica, "what I found interesting was that the CD4 cells were equally high for patients with high and low viral load if they had 'shifty 'viruses, whereas in patients with non-shifty viruses, the CD4 counts were low if the VL was high and vice versa, as you would expect. In the patients with the shifting virus populations, the high CD4 counts despite high virus load could reflect the 'discordance' that people like Stephen Deeks and the Swiss cohort have talked about. It also ties in with our data (also presented at Glasgow, and currently being reviewed by Annals of Internal Medicine) showing that clinical progression in patients receiving PIs who have high viral loads is significantly lower than patients not receiving PIs with equally high viral loads. In other words, we saw an independent treatment effect for PIs."
However, the responses to mega-HAART after TTI differed significantly between the WT-shifters and the non-shifters, as might be expected. The shifters experienced a -2.8 log10 drop in plasma viral load, whereas the non-shifters dropped just -1.02 log. After eight weeks of mega-HAART -- a very early time point -- 72% of shifters were beneath the limit of quantitation (BLQ), <500 HIV RNA copies/ml, versus just 10% of the non-shifters. Thus, the response to rescue therapy/mega-HAART was much better in the WT-shifters. Miller pointed out that because of these dramatic differences, both during the TTI and after the initiation of mega-HAART, viral load and CD4 counts need to be very closely monitored in this population.
Might there be immunologic differences between the shifters and the non-shifters? For example, did shifters have stronger specific anti-HIV responses, thus driving re-emergent viral isolates back to more "fit" wild-type virus, while non-shifters had such blunted specific anti-HIV responses that there was no such selective pressure. Veronica is interested in working with researchers who could use the new single cell interferon gamma release assay (such as Picker/Koup in Dallas or Roederer at Stanford) to quantify HIV-specific responses. San Diego's Diane Havlir assured her that these assays are possible when samples are shipped overnight on ice.
Another hypothesis would examine whether the resistant virus of the shifters was less fit than that of the non-shifters (some resistant viruses, such as those with NNRTI resistance, can be very durable, as shown by the MDR patient who transmitted an MDR, NNRTI resistant virus even though he'd been off nevirapine for over two years, suggesting no reduced fitness). John Mellors, clearly flabbergasted, asked how durable the virologic responses were in the shifters. Veronica answered that they looked good in the short term, but that follow up is not that far out. Some patients' virus remains suppressed after 11 months or more. "If it shifted rapidly, it wasn't very happy [fit] to begin with," she commented. "We're going to do fitness studies on the pre- and post-virus isolates." Towards the end of an animated Q&A period, ACTG co-chair Connie Benson -- echoing a similar caveat by Doug Richman in Chicago -- sternly warned that, however intriguing these results might be, they required careful further study in the context of controlled clinical trials with extensive laboratory analysis, and that "it would be wrong to encourage patients to do this on their own."