GMHC Treatment Issues, 9(10) - October 1995
Dave Gilden

This year's conference, which was held last month in San Francisco, counted the disappointments as much as the victories in the battle against pathogens. The theme was new and re-emergent disease.

AIDS is, of course, the archetypal new disease. It received plenty of attention, especially given the lack this year of an International Conference on AIDS (now held only biannually). In addition, people with AIDS are both the source and target of drug-resistant microbes. This double threat arises because their immune systems do not back up the therapeutic effect of drugs the way competent immune systems do. Drug-resistant germs have a greater opportunity to evolve in such an environment, which is precisely the environment in which the need for potent drug therapy is greatest.

In the following pages, we describe research presentations at ICAAC related to HIV and AIDS. The virus has turned out to be the most modern of bugs -- like the cockroach, HIV's high reproduction rate gives it a supreme capacity for mutating to evade the toxic chemicals attacking it. But, there has been at least incremental progress made in formulating strategies to circumvent this issue.

A More Prolific Virus...

At ICAAC, David Ho, M.D., director of the Aaron Diamond AIDS Research Center in New York, presented a continuation of his group's observations on the rapid pace of the HIV infectious cycle. (See these researchers' earlier observations in Nature, January 12, 1995, pages 123-6.) These results are based on following the blood levels of HIV and infected cells after patients begin receiving the powerful protease inhibitor ritonavir.

According to Dr. Ho's current estimates, at least ten billion HIV particles, on average, are produced each day in people infected with HIV. The lifespan of those free virions averages about eight hours, and the cells they infect live for an average of 2.2 days after infection. Dr. Ho calculated that the time between the creation of a new virus particle and the point that particle starts producing its own progeny within the cell it infects is about 1.2 days, making for about 300 replication cycles per year. These calculations show the high frequency with which we can expect mutations in the virus, which apparently undergoes the equivalent of thousands of generations of human evolution in the course of a single person's disease.

Dr. Ho claims that any single mutational change in any of the HIV genes probably occurs multiple times per day in a given human being, and forms of a single HIV gene with multiple mutations probably occur as well in the course of a day. Mutations alter the amino acid sequence and shape of an HIV enzyme or protein, and such alterations nullify the action of antiviral drugs or immune defenses that bind to and neutralize specific amino acid chains.

Steven Wolinsky, M.D., of Northwestern University, who gave one of the lectures at the ICAAC opening session on AIDS, pointed out that the ability to rapidly evolve may be the central reason why HIV ultimately defeats the immune system. The immune response to HIV depends on detecting specific portions of HIV's structural protein (epitopes) either on the virus itself or on the surface of HIV-infected cells.

Cytotoxic lymphocytes (CTLs) kill cells recognized as infected, and antibodies bind to their target regions on free virus particles and neutralize the virus. This activity creates selective pressure in favor of mutant HIV with proteins not targeted by existing CTLs and antibodies. The immune system may at first be able to engender new CTLs and antibodies to catch the escaping HIV, but eventually HIV comes up with so many diverse strains that an individual's immune system cannot take care of them all (the "diversity threshold" is exceeded). (Much of this material was described by Martin Nowak and Andrew McMichael in the June, 1995 Scientific American.)

The HIV evolutionary system that works to evade the natural hazards posed by the human immune system also functions efficiently to escape the perils posed by antiviral drugs that bind to the reverse transcriptase and protease enzymes. In the lecture preceding Dr. Wolinsky, John Coffin of Tufts University in Boston examined the rapid rate of appearance of resistance to our most powerful antiviral drugs.

The rapid appearance of resistance is testimony to the vast diversity of virus variants always present in the body, according to Dr. Coffin. He thinks mutant viruses with at least modest resistance to a given drug exist even before a person starts taking that medication. These mutants may not be detectable because they do not multiply quite as fast as the dominant "wild type" strain, but they rapidly takeover when HIV is exposed to a particular drug.

Dr. Coffin cited the example of 3TC. A mildly resistant mutant strain is indeed detectable before therapy, and then a more highly resistant single mutation shows up a few weeks after 3TC administration begins. "There is no way to hit the virus hard enough to prevent mutations," he concluded.

A final problem is that a few infected cells are chronically infected and do not naturally succumb at the rate Dr. Ho predicted. (These latently infected and/or long-lived, slow producers of HIV, probably macrophages and quiescent CD4 cells, are often termed "virus reservoirs.") Since present drugs only block HIV replication and cannot eliminate infected cells, there persists a small amount of virus production (0.1 to one percent of normal production even for the most powerful protease inhibitors). Virus reservoirs inescapably limit how hard any of the present medications can "hit" HIV over the short term, and mutant escape versions of HIV continue to have the opportunity to arise after drug therapy commences.

Dr. Ho calculates that the solution to the problems HIV variation poses is not necessarily powerful therapy, but more varied therapy. "We need combinations of drugs that force the virus to mutate at multiple points, preferably more than four, before the numbers favor the host," he said.

Previously, combination therapies have been promoted because of the drugs' additive or synergistic effects in reducing HIV replication. Dr. Ho is speaking about combination therapy from a different perspective, inhibiting the evolution of unwanted drug-resistant HIV strains by both reducing HIV replication and requiring an untenable number of mutations.

And a Proliferation of Resistance Factors

More and more researchers are accepting Dr. Ho's contention that the ultimate failure afflicting all current anti-HIV therapies is strictly associated with genetic mutations conferring drug resistance. And combination therapies seem a logical way to defeat HIV's talent for these mutations. A satellite conference held after ICAAC explored these two assumptions further.

The first lecturer, Daniel Kuritzkes, M.D., of the University of Colorado warned that all is not so straightforward. Citing two government-sponsored studies, ACTG 116B/117 and ACTG 192, Dr. Kuritzkes remarked that switching to ddI after AZT use apparently confers benefits regardless of whether a patient's HIV is still susceptible to AZT. More remarkably, the people with AZT-resistant virus do worse after switching than those with susceptible HIV. This is counter-intuitive: one would think that ddI would have the biggest impact in people with AZT-resistant HIV. They are the ones receiving the most minimal benefit from their present drug, and there is no evidence from laboratory cultures that AZT-resistance alone causes any decrease in susceptibility to ddI.

Fears have been raised for the past several years that some mutation in AZT-resistant HIV causes an increase in the virus' virulence. A similar, though less serious phenomenon is present during d4T and ddC therapy: CD4 count falls and disease progression do not seem related to the emergence of resistant strains. This is true, too, for saquinavir, Roche's protease inhibitor. According to the ICAAC report on using high dose saquinavir (ICAAC presentation LB-5, also see Treatment Issues, September, 1995, pages 6-7), HIV plasma levels bottom out and begin to rebound four weeks after starting the 7.2 gm/day dose, yet resistance mutations do not start to appear before the twentieth week of therapy.

One must remember all the factors that contribute to drug failure. The first of these is that since the currently approved HIV therapies are not really very good at stopping viral replication, they do not halt the progressive immunologic decline leading to AIDS. With less and less immune system backup, any drug will perform less and less well (this circumstance was described for CMV therapies in Treatment Issues July/August, 1995, page 1). Another factor is cells' increased ability to avoid metabolizing nucleoside analogs like AZT into their active, or phosphorylated, form (this "cellular resistance" was described by Michael Dudley in ICAAC presentation S110). Finally, the present drugs do not reach all the cell types harboring HIV. All these elements abet the rise of drug resistance by allowing further HIV replication and more mutations. The final outcome of the genetic rearrangement that evolves, as the virus attempts to optimize itself in the presence of drugs, is uncertain.

Although it is obviously important to look at the whole environment in which HIV and drugs interact, an elegant report by Charles Boucher, M.D., at the satellite resistance conference, showed how well merely following the HIV genetic response to chemical therapy can explain the observed results.

Dr. Boucher and his associates looked at a group of trial participants in Amsterdam who first received 3TC monotherapy, to which AZT was later added. The unique, rapidly appearing mutant that creates 3TC resistance is supposed to block the effects of the AZT resistance mutations, but Dr. Boucher says that this is only true for the initial alteration of the reverse transcriptase that causes low-level AZT resistance. The succeeding mutations, which result in high-level resistance (and possibly greater viral virulence), are little affected.

In the Amsterdam 3TC-resistant patients, high-level AZT resistance occurred later than usual, in the second year of AZT/3TC therapy, via several of the usual mutations plus a novel one.

The ability of high resistance to AZT to coexist with 3TC resistance suggests that it will not help so much to add 3TC to the regimens of people with lengthy experience on AZT (in whom the mature AZT-resistant genetic pattern is frequent). And indeed, the long-term observed antiviral effect in clinical trials of adding 3TC to prior AZT monotherapy was comparable to adding ddC. (See Treatment Issues, February, 1995, pages 3-4.)

Dr. Boucher nevertheless held out the hope that all these mutations will render reverse transcriptase a less functional enzyme, and the HIV producing it will replicate less well. The extent to which this holds true, as well as the physical benefit patients receive, will have to be determined in clinical trials. A previous attempt to create "replication- deficient" HIV -- through the so-called convergent combination therapy -- was not very successful (see Treatment Issues, January, 1995, page 6).

Beyond the Valley of the Drugs

A combination therapy that includes AZT, ddC and Abbott Laboratories' protease inhibitor ritonavir is performing significantly better than any past regimen, with HIV levels continuing to decrease in a handful of patients, so far, out beyond at least five months (see page 6). An ongoing trial testing AZT plus 3TC and indinavir (the Merck protease inhibitor) may show that this combination performs as well as the ritonavir-containing one.

Abbott investigators are already talking about "draining the reservoirs" of HIV. It is quite a leap to think that this type of combination can completely clear the body of HIV, though. One immediate problem is that protease inhibitors penetrate the brain poorly if at all. Most likely, people with HIV will be required to take an effective drug therapy absolutely every day for the rest of their lives, regardless of side effects. If therapy is missed for just a few days, HIV replication will start to return, with the heightened possibility of a virus evolving that is both virulent and drug-resistant. This scenario is similar to the one that exists for tuberculosis, only worse.

We still have a hard time controlling TB, and drug-resistant tuberculosis is becoming a major problem. For ultimate HIV control, then, maybe we will have to hit the virus not just "hard," but from different directions. Hydroxyurea, as reported in our last edition, and other compounds that make the cellular milieu less compatible to HIV, offer a variety of new approaches. Compounds that break HIV's "zinc fingers" are also a possibility here (see page 7).

Immune-based therapies, which work by ameliorating the immune response, could be the final answer since they would work on the heart of the problem -- the lack of immune backup to chemotherapy. Unfortunately, the initial attempts have not worked well. IL-2 (see Treatment Issues, February, 1995, pages 7-11), for example, failed to lower HIV levels over twelve months of treatment although, as in the past, it did double CD4 counts (ICAAC presentation LB-8). No clinical benefit from IL-2 has yet been demonstrated, either.

Another approach has been the so-called therapeutic vaccine, in which synthesized HIV proteins or killed HIV stripped of its outer coat has been injected into people with HIV in the hope of coaxing a broader immune defense that reacts to more critical HIV epitopes. Like IL-2, these vaccines have had no confirmed benefit in terms of HIV levels or patients' health.

There were two early reports at ICAAC on the most sophisticated therapeutic vaccine yet, Viagene's gene therapy product which induces cells in the body to mimic HIV infection by producing HIV proteins. The presenters claimed improvements in immune response, but detected no significant change in HIV levels or CD4 counts (presentations I75 and I76).

An alternative way of modulating the immune response to HIV would be to reduce aspects of that response that are considered counterproductive. Use of thalidomide to inhibit the inflammatory cytokine and HIV-stimulator tumor necrosis factor (TNF) has been widely discussed (see Treatment Issues, May 1995, pages 3-6). At ICAAC, one report described using a fusion protein including part of cells' TNF receptor to "mop up" excess TNF. A pilot study of this molecule again observed no effect on HIV levels or CD4 count (presentation I78).

Nevertheless, immune-based therapy seems like an important avenue to pursue at this point. "It's clear that antiretroviral therapy has been limited by a number of factors that are quite straightforward. It might make sense to refocus our thinking on the virus-host interaction," contended Robert Schooley, M.D., of the University of Colorado, during a review of immune-based therapies (presentation S51).

We need to identify the responses that keep HIV in check early in disease and in long-term nonprogressors. A successful immune-based therapy would then reinforce or recreate such responses in the human body. As drug therapy keeps HIV levels low, a powerful and properly cultivated immune response could seek out and eliminate the last vestiges of infection wherever they may be.

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