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Infectious HIV Persists After Up to 2 1/2 Years of Triple Drug Therapy, Due to a Non-Decaying 3rd Compartment

Its the immune system after all.

Can HIV be eradicated from a human host? Can currently available combination therapy approaches eliminate HIV from every last cell within a reasonable amount of time (short of the lifetime of the host)? This provocative question was first raised before a global audience by David Ho at Vancouver in July 1996. After months of rumors and an accumulation of presentations at St. Petersburg in June, at Istanbul and Toronto in September, and at Hamburg in October, three papers published in Science and the Proceedings of the National Academy of Sciences (U.S.A.) in November 1997 definitively laid to rest the idea that HIV could be eradicated within three years using currently available triple combination antiretroviral therapy. Mark Harrington helps us make sense of it all with this sobering and long feared year-end report.

The problem is that current combination therapies, no matter how potent, only act against the virus when it is replicating. If there is a significant number of cells in which the virus is resting dormantly — integrated into the nucleus — the drugs will be unable to target these infected cells which, when activated, will be able to reignite the infection — no matter how long someone has suppressed new virus production with combination therapy. Thus, the last sixteen months have seen an intense search for a cell population — the so-called “third compartment” — in which HIV might persist in dormancy, unresponsive to combination therapy. These experiments built on work first shown by David Ho at Vancouver in July 1996, and followed up by Ho and Los Alamos Alan Perelson at the Fourth Retrovirus Conference in January 1997, which estimated the time it might take to eradicate HIV from an infected human host. These were the first rational, quantitative estimates based on in vivo data on viral and host cell kinetics, and they assumed that there were just two cellular compartments in which HIV dwelled:

  1. A primary compartment consisting of activated, HIV-infected CD4 T cells actively producing virus and rapidly being killed either by HIV or by the immune response, which turn over rapidly in the body with a half-life of a 1.4 days, and produce over 90% of the new virus population;
  2. A second compartment — possibly consisting of latently infected CD4 T lymphocytes or macrophages — which, when activated, produce virus, turning over with a slower half-life of one to four weeks, and producing less than 10% of the new virus population.

Regardless of the number of HIV-infected cells in the body, if these were the only two compartments in which HIV dwelt, the time-to-eradication could be estimated. If the half-life of the second compartment was four weeks, the time-to-eradication would range from two to three years, depending on the number of HIV-infected cells. The entire model, however, would break down if there proved to be a third compartment. If one existed, it would be important to measure whether it, like the first two compartments, was reduced in size by triple combination antiretroviral therapy and, if so, by how much, and how fast. Only then could one reliably estimate the time-to-eradication using current therapies. Now, it turns out that there is a third compartment. It consists of latently infected, resting, previously activated memory CD4 T lymphocytes with integrated proviral HIV DNA in their nucleus. These cells are a small but crucial reservoir for HIV and, at least after two years of triple therapy, do not appear to diminish over time.

In other words, the third compartment does not have a half-life of decay, because it does not appear to decay at all. If this holds true, then we can no longer expect the current therapeutic approaches by themselves to lead to HIVs eradication — which is really just a fancy word for cure. New approaches, however, could be developed to target this third compartment, possibly speeding up the turnover of these cells and leading to their elimination. Such approaches remain speculative, but some are rapidly moving towards preliminary clinical trials. In the meantime, however, no one should begin antiretroviral therapy with the belief that triple (or even quadruple) antiretroviral therapy is likely to eradicate HIV infection. The best we can hope for, at this time, is chronic suppression of HIV and thereby preventing disease progression, protecting the immune system from further damage, and avoiding the development of drug resistant HIV strains.

HIV Infection, Reverse Transcription, CD4 Cell Activation, DNA Integration

Most HIV in the body is in the form of RNA, either as free virus particles circulating in the blood, trapped within lymph nodes or inside cells either just after invading a cell (before it is converted into DNA) or when an infected cells HIV DNA is making new viruses and new viral RNA. Each infected cell may produce from 1-200 new virions. The reservoir which accounts for the persistence of HIV infection is, however, not viral RNA but proviral DNA which has already been integrated into the cell nucleus.

HIV DNA can be detected either in an unintegrated form within the cell cytoplasm, or integrated within the cells DNA in the nucleus. Robert Siliciano, of Johns Hopkins University, and colleagues developed an assay earlier this year to distinguish the two types of HIV DNA. They found that if a cell is infected while in a resting state, reverse transcription may occur, but the new HIV DNA will not be transported into the nucleus for integration. If the cell is kept in a resting state for six days, cellular proteins will degrade the HIV DNA and integration will be aborted. Thus, cellular activation is required for integration and productive infection of a cell.

Most cells which experience activation and integration then undergo mitosis (cell division), in the process of which HIV copies are made. The cell is either killed by direct cytolytic effects of the virus or by antiviral effector mechanisms of the immune system (cytotoxic T lymphocytes (“CTLs”), antibodies, macrophages). However, a small minority of infected cells may survive with integrated HIV DNA. Of these, only a small subset are replication competent. Of one million white blood cells taken from an infected individual, 1,000-100,000 cells may have detectable HIV DNA provirus, but only 10-100 of these cells will have integrated provirus and only 1-10 of these will be replication competent. (Other cells, however, may remain targets for the immune mechanisms mentioned above if any viral proteins, even defective ones, are made.)

Discovery of the Third Compartment

Resting memory (CD45RO+) cells are the major reservoir for integrated HIV-1 provirus in resting CD4 T cells. Three studies (Finzi/Siliciano, Spina/Richman and Chun/Fauci, 1997) show that there is no apparent decrease in the number of latently-infected resting CD4 cells over two to 32 months of potent antiretroviral therapy. Even among individuals with fewer than 200 RNA copies/mL, one to ten infectious units per million cells (IUPM) can be detected after up to 32 months of treatment. The assay is imprecise (with up to one log variation), but this suggests that there may be a very slow half-life for decay of this infected cell population, confirming estimates made several years ago by Angela McLean of Oxford University.

All three teams took a similar approach in searching for the resting, latently infected CD4 cells. First they eliminated B cells, macrophages, natural killer cells and CD8 cells. Activated CD4 cells were eliminated to make sure the sample cells were truly resting. Then they stimulated the resting T cells into activation thereby “turning on” any replication-competent HIV DNA which may reside within this cell population. They measured HIV within the activated, previously resting cells in several ways: by looking for proviral DNA in the nucleus, or unintegrated DNA in the cytoplasm; by amplifying proviral DNA with PCR; and — importantly — by culturing the cells to see if replication-competent HIV could be grown from the cells. It could.

In the largest study, led by Silicianos team, Finzi and colleagues examined cells from 22 individuals treated with tripe therapy for up to 30 months. Subjects had to 1) be on highly active antiretroviral therapy (“HAART”); 2) be highly adherent; 3) rapidly have achieved “undetectable” plasma HIV RNA levels (<200 RNA copies/mL by RT-PCR); and 4) have remained “undetectable” for the duration of the study. Replicating virus was detected in all 18 cases when sufficient resting CD4 cells were isolated (they could not be isolated from four individuals with low CD4 counts). Very few resting cells had integrated HIV DNA which could be stimulated into active replication — just 0.2 to 16.4 infectious units per million resting cells. But what proved to be the most discouraging finding is that when, in a cross-sectional analysis, the number of infectious cells in individual patients were correlated with their time on HAART, there appeared to be no relationship between time on HAART and number of infectious cells — and thus no apparent decay in the third compartment. Serial samples were taken from only one patient — whose level of infectious cells actually rose slightly between months four and eight. Thus, while the third compartment appears to be small, it also appears to be very stable.

To see how old the provirus in these resting cells was — and whether it had evolved resistance to combination therapy — the investigators sequenced the protease genes. The good news is that (in patients with plasma RNA levels continually suppressed below 200 copies/mL), the proviral HIV did not display mutations associated with drug resistance. This indicates that the cells had likely been infected prior to the initiation of triple therapy, and that viral evolution appeared to have greatly slowed down — if not stopped altogether — after the initiation of triple therapy. (Some individuals had mutations from previous experience with monotherapy.) “Taken together,” the authors explain, “these results demonstrate that purified resting CD4+ T cell populations from patients on HAART harbor replication-competent virus that in some cases is cytopathic.” (Given the inherent vagaries of translating results from in vitro experiments to in vivo conditions, however, the question of whether these latently infected T cells can be reactivated to produce virus in treated individuals remains, for now, unanswerable. GMHCs treatment wonk Dave Gilden, writes in a second exhaustive analysis (Treatment Issues, November 97) of the 3 eradication papers, “It is not clear, though, that any particular therapy is needed for latent cells. It is possible that as the immune system recovers in the nearly HIV-free environment, anti-HIV cytotoxic lymphocytes will be able to quickly kill the few cells with active HIV infection and prevent spread of the virus;” to which Diamonds Ho replies matter-of-factly, “I wouldnt bet on it.”) The Johns Hopkins investigators are understandably wary to come down on either side of this issue and warn only that “the existence of a small but relatively stable compartment of latently infected cells should be considered in deciding whether treatment should be stopped in patients with no other evidence of residual virus.”

In the second paper, Joseph Wong, Doug Richman and colleagues conducted a similar but smaller study at U.C. San Diego. They isolated resting CD4 T lymphocytes from six individuals who had achieved undetectable (<50 copies/mL) viral load for up to two years on HAART. As in the previous study, infrequent cells with integrated proviral HIV DNA were found, their protease genes sequenced, and no genotypic evidence of protease resistance identified, suggesting that the third compartment is stable over up to two years of HAART and that HAART prevents viral evolution and the development of drug resistance. In summary, there is evidence for a third phase of viral persistence among latently infected CD4 cells with integrated provirus. These cells are predominantly CD45RO+, and their isolation requires CD8 cell depletion and CD4 cell activation.

The half-life of this third compartment is likely to be many months to years, but the virus isolated from these cells taken from patients with very low HIV RNA levels (<50/mL) after two years of treatment showed no evidence of viral evolution or the emergence of drug resistance. Thus, hopes for short term eradication of HIV from an individual within three to six years are “clearly unrealistic,” according to Richman, unless we can figure out a way to shorten the third phase, perhaps by stimulating the latent infection out of the CD4 cells. “Conceivably,” he continued, “one could eradicate in ten years or more.” Richman joked that inducing toxic shock might be one way of shortening the third phase and concluded that, “It would be wrong to be discouraged about anything but the prospect for short term eradication; we have achieved suppression of viral evolution, which bodes well for maintenance.”

The third paper, from Faucis lab published by Chun et al. in P.N.A.S., also found persistent low level RNA production despite triple therapy. Eighteen HIV-infected individuals on various antiretroviral regimens had leukopheresis (blood extraction) at the NIH, and their cells were analyzed for HIV RNA at baseline and, in thirteen patients, after a median of ten months of “HAART.” They were able to isolate resting memory CD4 cells with integrated, replication-competent HIV provirus at low frequencies from 11 of the 13 patients on HAART. Some of these viruses (in cell culture) could induce syncytia, indicating virulent cytopathicity. They also found low levels of unintegrated DNA in some cells, indicating recent infection and reverse transcription. The amount of unintegrated DNA was higher in patients whose viral load was still detectable. In contrast to the Siliciano and Richman paper, the NIAID study “suggests persistent active virus replication in vivo,” but this might simply reflect the fact that viral suppression in the NIAID study was less stringent (“undetectable” in the NIAID study was defined as <500 copies/ml) than in the other two studies (Siliciano, <200; Richman, <50/mL).

In addition, the NIAID team found that “levels of integrated HIV-1 DNA within resting CD4+ T cells in HAART naïve patients was not significantly higher… than in treated patients… [suggesting] that resting CD4+ T cells with integrated HIV-1 DNA do not decay rapidly in patients receiving HAART and thus represent a stable reservoir of HIV-1 DNA.” In other words, the third compartment appears to lack a measurable decay curve despite potent combination antiretroviral therapy. Chun et al. concluded their paper by noting that “the time required for virus eradication — if indeed this is possible — will be considerably longer than previously predicted,” and they call for the development of “strategies for eradicating those minor populations of infected cells that serve as reservoirs of inducible and replication-competent HIV.”

What Can Be Done to Eliminate the Third Compartment?

Several strategies are currently being considered to eliminate the third compartment. One way might be to activate the latently-infected resting cells so that potent antiretroviral combination therapy would interrupt further cycles of virus replication. There are several obvious dangers to this approach. First, the amount of virus that might be released might be enough to overcome therapeutic barriers against the development of drug resistance. Second, the activation of so many resting CD4 cells might cause excess inflammation, fever or even shock.

ACTG 387 is a protocol in development which aims to determine more exactly the kinetics of viral decay with the three current classes of antiretroviral agents (nucleoside and non-nucleoside RTIs and protease inhibitors) by measuring their pharmacokinetics and viral kinetics in the first 72 hours of therapy. Participants will then begin four drug therapy with AZT, 3TC, indinavir and nevirapine. After six days they will be randomized to receive immune modulation with interleukin-12 (IL-12), GM-CSF, or nothing. IL-12 is a Th1-type cytokine which induces production of gamma interferon, and may inhibit CD4 cell apoptosis. Immune modulation will continue for fourteen weeks and quadruple antiretroviral therapy will continue for 48 weeks. The hypothesis here is that IL-12 will accelerate the clearance of HIV from long-lived CD4 cells and that GM-CSF will do the same for macrophages. This study may help to clarify whether it is possible to use immune stimulatory cytokines to speed up clearance of the third compartment.

Additional Questions

It seems clear that, with maximal antiretroviral control of active HIV replication, weve gotten as far as we can with “Its the virus, stupid.” Many of the most pertinent questions now relate to the immune system and its ability (or inability) to contain HIV infection:

  1. Can memory cells revert to naïve cells in humans? How frequent is this?
  2. Can latently infected cells undergo mitosis into infected daughter cells without turning on viral replication?
  3. Do memory cells that revert to naïve cells retain their capacity for further T cell receptor gene reshuffling?
  4. Is there new thymic emigration in HIV-infected adults?
  5. Is there extrathymic maturation of new naïve or memory CD4 cells?
  6. As the likelihood of eradication recedes, does the argument for treatment of newly infected or asymptomatic individuals weaken?
  7. What destroys the lymphoid tissue late in disease?

Some of us posed the last question six years ago. We still dont have the answer.

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