TAGline/Volume 4 Issue 2

1. Druthers, Grim: Frightening Tales of Multiply Resistant HIV from D.C. Principles Panel
2. Backstepped: Dutch Research Team Challenges Ho/Shaw Pathogenetic HIV Model
3. Staring into the Precipice: resistance and cross-resistance among protease inhibitors

The NIH panel convened late last November to develop new state-of-the-art guidelines for the treatment of HIV infection included a series of provocative (some would say alarming) presentations regarding resistance considerations in the choice of antiretroviral combination regimens. TAG's indefatigable scribe and provocateur in his own right, Mark Harrington, provided this transcript of the cross-fire which featured, among others: Merck's Emilio Emini, Tufts University's John Coffin, University of Montréal's Mark Wainberg, Roche's Noel Roberts, the CDC's Harold Jaffe, Chiron's David Chernoff, the ACTG's Robert ("Chip") Schooley and John Mellors, as well as fellow treatment activist Dawn Averitt-Doherty of Atlanta-based Woman's Information Service and Exchange (WISE).

Mark Wainberg presented interesting resistance data from BI 1046 (the INCAS study of AZT+ddI+nevirapine (NVP) vs. AZT+ddI vs. AZT+NVP in treatment naïve individuals with CD4 cell counts 200-600), analyzing compliance as defined by any patient who did not miss more than 28 days of any therapy. On triple drug therapy, viral load was reduced by 1.5 logs at 23 weeks. Patients non-compliant to ddI rapidly became resistant to nevirapine. At twelve months, even some of those who were compliant developed NVP resistance, although in most patients on triple therapy (87%) it was difficult to culture virus. At twelve months, virus could not be cultured from any patient whose viral load was beneath the limit of detection (LOD). When treatment reduces viral load below LOD, virus culturable from peripheral blood mononuclear cells (PBMCs) at six months is wild-type. When treatment fails, virus culturable from PBMCs as six months is NVP resistant, but not as frequently (or as highly so) as in patients receiving only AZT and NVP.

Coffin: Both studies (BI 1046 and the Merck 035 study, presented earlier that day) raise the question of whether there's a rationale to include AZT anymore. It makes no difference between experienced and naïve patients. NVP and AZT didn't push the virus very hard.

Emini: The combination of d4T and 3TC should be looked at.

Coffin: We need to study the fitness of various mutants in the absence of therapy. Some mutants will have a high cost to fitness, and will be infrequent; while others may have a low cost and may be more common. There may be an evolutionary bottleneck early in disease before which such pre-existent mutants are rare, but six months into infection-or after a year-you've lost that advantage.

Harrington: It seems to me there are two ways to avoid the development of resistance here: one is to suppress maximally with potent antiretroviral regimens. The other is to wait to begin therapy until it's necessary to prevent irreversible immunologic damage. If the only point at which to throttle the virus from an evolutionary standpoint [to limit the pool of pre-existing drug-resistant viral mutants] is in the first 6-12 months of infection, this has little relevance to most chronically-infected patients who, given the limitations of our arsenal, might be better of waiting for a clear need to emerge.

Roche's Noel Roberts presented cross-resistance data on clinical isolates which had been presented at the Birmingham conference, suggesting that HIV that has developed resistance to any of the three licensed protease inhibitors is likely to be resistant to the other ones (licensed and unlicensed) as well, and that resistance to IDV (currently the most widely prescribed protease inhibitor) appeared to confer 60% cross-resistance to SQV, 80% cross-resistance to the Vertex compound (141WU94) and 100% cross-resistance to RTV and NFV! University of North Caro-lina's Robert Swanstrom took it a step further and looked at HIV that had become doubly protease resistant (to SQV+RTV, RTV+IDV and SQV+IDV). In his in vitro experiments, HIV that was doubly resistant to IDV+RTV was 400-fold less susceptible to SQV. And HIV with dual resistance to SQV+RTV was 60-fold less sensitive to IDV. So the implication for people failing SQV, IDV or RTV appear grim-at least from this data.

Schooley: We need to be pretty careful and do in vivo studies of switching people from saquinavir to other protease inhibitors and vice versa.

Roberts: That study is underway.

Chernoff: In these trials, drug is continued post-failure, unlike in clinical practice. [This is not true; many doctors continue patients on therapy post-failure. What else can they do? There are no clinical practice guidelines out there, and many people have failed all available drugs.]

Roberts: Perhaps patients should change therapy as soon as viral load starts to rise.

Mellors: It troubles me to hear a comparison of the frequency of resistance without controlling for the potency of the various regimens. The two variables are related-viral turnover and the selection process imposed by more or less potent regimens.

Jaffe: If resistant mutations are associated with a selective disadvantage will they disappear after treatment is removed?

Emini: Two patients who stopped taking indinavir (one after taking low-dose, then full-dose) had a reversion to wild-type within 4-5 months post-cessation. One restarted treatment at full-dose idv, and within three weeks we isolated idv-resistant virus. This is why we say the virus is "genetically unforgiving."

During the coffee break, I joined three activists outside to share nicotine and despair. What was the point of quitting smoking if we were still all passengers on the speeding train heading for the cliff? The Birmingham resistance data were wrenching. Our fears of multiple cross-resistance, from November 1995's 3TC and saquinavir FDA approval hearings, reared their ugly heads. Several months of post-Vancouver euphoria crumbled in a moment as it became clear that many of those who developed resistance to ritonavir and idv-as thousands clearly would-might have no protease inhibiting options ahead of them. Today's resistance news made for a toxic cocktail. As I left the auditorium I bumped into Emilio Emini.

Harrington: So what do you do if you fail Crixivan?

Emini: [sighs] We don't know what to do.

Harrington: Take two new nucleosides and nevirapine?

Emini: Yeah. And pray.

No one had yet assessed the healing effects of prayer on viral load. This was what we'd come to. I rushed into the lobby of the Interior Department and ran into a colleague, who was wild with fear and disappointment.

AC: I'm going to die.

Harrington: This is everything we were hoping wouldn't happen.

AC: I don't have anything to switch to. I'm going to stop everything.

Dawn Averitt tried to calm our colleague down: Why don't you wait for you next viral load?

AC: I'm already back at baseline!

Averitt: But your CD4 count is 250, and it was down to 60 in January.

AC: True, but my viral load is back to half a million.

Averitt: Why don't you wait until you get your latest numbers and see what your resistance profile is?

AC: I'm going to go outside and have a nervous breakdown.

We went outside. It was frigid, and fragile snowflakes swirled around in the wind. Sometimes the gap between how the researchers felt and how we felt became an abyss. They were excited about the endless possibilities opened up by the research advances of 1996; we were terrified about the limited treatment options facing people who had exhausted most of the current arsenal of antiretroviral therapy. What to do with those whose viral load refused to go undetectable? What to do with those who added a protease inhibitor to a failing two-drug regimen and appeared doomed to develop resistance, most of it-especially with ritonavir and idv-cross-resistant to all other protease inhibitors? What to do with those who jumped aboard last year's bandwagon, AZT+3TC, and now appeared likely to have developed 3TC resistance and, with it, cross-resistance to ddI, ddC and possibly 1592? The Chinese menu approach to antiretroviral treatment sud denly looked much less appetizing, and much less nourishing.

Dawn and I went upstairs where the committee was having a working lunch, discussing process. Many members questioned the existence of two committees. Why have one committee setting up principles (NIH) and one setting up practice guidelines (PHS)? Wasn't this a recipe for bureaucratic confusion-and delay? How could we disseminate principles of HIV therapy without making practice guidelines? What if the principles contradicted the data? Considering what we had just seen, doing something fast seemed imperative.

On the other hand, many of the researchers present did not share the activist sense that we were facing a crisis that, if handled improperly, might make things worse than before. This was not the prevailing view propounded by gun-happy virologists, drug-happy pharmaceutical companies, media captivated by a surprising good-news story and many people with HIV still struggling to absorb the complex developments of 1996. Just that week, back-to-back articles in The Wall Street Journal and The New York Times Magazine, both written by HIV-infected journalists, declared that the epidemic was virtually over. We were staring into the precipice while others were still climbing the hill.

As long ago as 1995, when the back-to-back Ho and Shaw viral dynamics papers were published, HIV disease has been likened to a colossal bathtub-or sink rather. The thinking went like this: the pool of CD4+ T lymphocytes ("CD4 cells") coursing through your body can be thought of as a sink of water. And like most sinks, this sink has both a faucet (or "tap") and a drain. Open the tap, and the level of water in the sink begins to rise; open the drain, and water rushes out. Pretty basic, TAG's Gregg Gonsalves explains.

Dr. Ho began using this analogy for what happens to the body's population of CD4 cells throughout the course of HIV infection. According to the now nearly universally accepted "sink" model, a normally functioning immune system consists of a tap from which constantly flows a moderate volume of new T-cells (to replace old ones as they naturally die, self-destruct or senesce) and a drain through which trickles only a small number of old and non-functional T cells. In HIV disease, however, the drain is wide open, and CD4 cells, as a result of direct killing of cells by the virus, immune system killing of virus infected cells, cell suicide or, most likely, a combination of these (and quite possible other) events, are being sucked out the drain like street soiled rainwater down a storm sewer during a midsummer downpour. Even were the body's immune system to try to compensate (which it appears to) for the accelerated loss of CD4 cells by opening the tap to full throttle, it still could not quite keep up with the action down there at the drain-and the level of "water" (the body's CD4 cell count) gradually falls until the sink holds but a shallow puddle. "Plug up the drain," the sink proponents argue, and you'll stem the progressive immune deficiency. As a result, the vast majority of therapeutic approaches to HIV infection have focused on "plugging up the drain," that is, halting the destruction of CD4 cells by stopping the virus.

November 1996: The Third International Congress on Drug Therapy for HIV Infection was held in Birmingham, England. A team of hotshot young virologists from the University of Amsterdam and the Amsterdam Medical Center (among them Frank Miedema, Jaap Goudsmit, Jaap Lange, Sven Danner) presented data suggesting that this "sink" model of HIV infection is all wrong. Rather than AIDS being caused by exhaustion of the regeneration capacity of the immune system as a result of high T cell turnover (the "drain"), the researchers claimed, AIDS is caused instead by interference with T cell renewal (the "tap"). At the very foundation of their blasphemous assertion was work done with hitherto obscure bits of protein call "telomeres."

Telomeres are lengthy stretches of contiguous, repeated simple DNA sequences (in vertebrates: TTAGGG x n) at the very end of chromosomes which play an important role in maintaining chromosomal integrity. When a cell divides, one end of the cell's chromosomes never gets fully replicated; the enzymes responsible for copying the chromosomes for the new cell always stop just short of the end of the line. So telomeres serve as a sort of genetic padding at these ends-padding that can be left behind without detriment to the cell as it divides. This ensures that no important genetic information is lost in the transfer to the next generation of cells.

In humans, telomeres are initially 10 kilobases in length. After about 25 cell divisions (which generally takes decades in a normally functioning human body, at a loss of 30-50 base pairs per year for normal human cells), cells can no longer divide and enter into an irreversible state of growth arrest known as "replicative senescence." By this point, telomere lengths have shortened to a mere 5-7 kilobases. Although somewhat esoteric, this phenomenon comes in quite handy: by looking at the shortening of telomere lengths over time one can assess both the replicative history and the proliferative potential of the cells in question.

With this as a backdrop, one of the obvious questions in the search to understand how HIV infection leads to immune system collapse in humans is whether or not the high rate of CD4+ cell turnover first reported by David Ho and George Shaw's laboratories is also accompanied by telomere shortening. It is exactly this question which Miedema and his Dutch colleagues presume to have answered. First presented at the Birmingham meeting, and then published shortly thereafter in the November 29 issue of Science magazine, the Dutch team concluded that, "Our data do not support the idea of high rates of production and destruction of CD4+ T cells as depicted in the 'sink model' proposed by Ho et al." Rather, "we suggest that HIV-1 infection is slowing down the flow of the tap, that is, the generation of new cells from an as yet undefined precursor source." Of interest, however, is the fact that Miedema did find shortened telomere lengths in CD8+ cells from HIV-infected individuals. This confirms a recent study by Janis Giorgi of UCLA, who reported last July that the CD8 telomere length in HIV-positive persons approximates that of healthy centenarians (AIDS 1996;10:F17-22).

The Dutch investigators hypothesize that HIV infection may interfere with the generation of CD4+ T-cells "through (i) the infection of stromal cells or microenvironment damage or both; or (ii) by infection of dividing CD4+ cell precursors, which would therefore abort the influx of new cells." Stromal cells are large, spread out cells that provide a bed for many blood cells; in this case, developing T-cells in the thymus. Thymic stromal cells play an important role in T-cell maturation. Their infection or the destruction of the thymic microenvironment where they reside-and where T-cells develop-would have disastrous consequences for the generation of T-cells. Additionally, if T-cells, before they mature into CD4+ or CD8+ cells from CD4+CD8+ precursors, were infected and killed by HIV, there would also be a crisis in the influx of new T-lymphocytes.

Miedema and his collaborators realize that there is not a lot of experimental evidence for their hypothesis. In fact, their data suggesting that CD4+ cells are not turning over rapidly-but that CD8+ cells are-belies a hypothesis that depends on destruction of CD4+CD8+ precursor cells. Miedema et al. propose that the increase in CD4+ cell counts after treatment with potent antiretroviral regimens may not be due to repopulation with newly generated cells, but effected by redistribution ("retrafficking") of activated memory CD4+ cells instead. This mechanism of CD4+ repopulation is seen after ablative cancer chemotherapy; the only difference being that following cancer chemotherapy CD4+ cell replenishment requires a full 10 months or so-and all the newly generated CD4+ cells are CD45RA+, that is, of a naïve phenotype.

Needless to say, the Dutch paper has generated a great deal of controversy. Miedema's own admission of a lack of evidence to support their provocative conclusions is mirrored in the skepticism of other investigators about the interpretation the Amsterdam investigators have derived from their data. By comparison, the Ho-Shaw "sink" model has a great deal of data to back it up. Many groups have shown that there is a high level of activation in the T-cell compartment-and activated cells are very likely to be dividing.

Ho et al. have shown that there are billions of cells at any given time expressing cell surface proteins associated with cell proliferation (ki67). Miedema's own group has shown that there are a large number of cells dying as a result of programmed cell death (apoptosis). To maintain stable CD4+ cell numbers over the short term (as is generally seen in the intermediate years of HIV infection), rudimentary mathematics dictate that there must also be a comparable influx of new cells. Miedema and his colleagues fail to address this apparent contradiction.

As for the ADARC group's response to the Miedema challenge, Diamond's Bill Paxton notes that the heretical proposition of Miedema et al. is but one possible explanation for what's going on. "At the end of the day," Paxton advises, that paper is open to interpretation." New studies which radioactively tag cells from SIV-infected and uninfected monkeys may help to resolve this important debate.