TREATMENT OF RESIDUAL HIV-1 DISEASE: PURGING LATENT

HIV-1 CELLULAR RESERVOIRS AND THE REQUIREMENT FOR INDUCING DISTINCT VIRAL ARCHIVES FROM LATENCY

Stimulatory Therapy

The treatment options for attempting to rid the body of cells infected with HIV-1, which can produce replication-competent virus in patients on effective HAART, are now beginning to be explored based on our increased understanding of molecular lentiviral pathogenesis.

Some patients on suppressive HAART treated with intermittent IL-2 had decreased or unde­tectable levels of resting CD4⁺ T cells containing replication-competent virus.⁸⁷ When treatment was interrupted in two patients in whom replication-competent virus could not be found in resting CD4⁺ T cells, virus was readily isolated in vitro, and high levels of viral RNA in plasma were observed within weeks of stopping therapy.⁸⁹ Another larger group of 18 patients with undetectable levels of virus for more than 1 year had therapy interrupted.⁹⁰ All patients relapsed to relatively high levels of plasma HIV-1 RNA in 2 to 3 weeks after stopping HAART.

Other cytokines, such as IL-7 and -15, were proposed not only to activate latently infected T cells and increase the turnover rate of the latent viral reservoir to promote viral clearance but also as an immune-adjunctive therapy to HAART.

IL-7 and -15 have pleiotropic effects on T cell homeostasis. IL-7 enhances mature T cell survival,^91-93 expansion of both peripheral CD4⁺ and CD8⁺ T cells in T cell-depleted hosts,⁹⁴ and mobilization of pluripotent hematopoietic stem cells from the bone marrow to the peripheral circulation.⁹⁵ Importantly, IL-7 improves both HIV-1-specific CD8⁺ cytotoxic cellular activity in vitro in humans⁹⁶ and CD4⁺ Th cell-dependent humoral responses and CD8 cytotoxic T cell activity in mice immunized with HIV-1 envelope protein.⁹⁷ It was reported that in patients with AIDS, the plasma levels of IL-7 and -15 were higher than in healthy donors and that increased circulating IL-7 levels in HIV-1-infected subjects were inversely correlated with CD4⁺ T cell loss and positively correlated with plasma HIV-1 viral load.⁹⁸ This probably represents a homeostatic response to lymphopenia, because the high levels of IL-7 decrease when the T cell count increases after a HAART regimen is initiated.⁹⁸ The authors hypothesized that IL-7 might play an important role in HIV-1 disease progression.

Of note, IL-7 was shown to be able to induce HIV-1 replication in vitro from PBMCs of HIV-1-infected individuals who were not on HAART^99-101 and to increase the expression of HIV-1 Tat mRNA in CD8-depleted PBMCs from HIV-1 chronically infected patients.⁹⁹ Moreover, recent in vitro studies have indicated that IL-7 has minimal effects on T cell phenotype, whereas it induces substantial expression of latent HIV-1,^102-104 which suggests that IL-7 may be used as an additive therapy to HAART, not only for its immunologic effects but also to purge HIV-1 latent reservoirs.

Intensification Therapy

Some current clinical strategies approach the challenge of eliminating low levels of ongoing replication-competent virus through an “intensification” protocol that relies on administration of additional viral inhibitors apart from the combination of drugs that typically constitute HAART. Candidate drugs used in intensification with HAART include hydroxyurea (HU), although this should only be used in intensification research protocols based on its toxicity, especially in com­bination with the reverse transcriptase (RT) inhibitor didanosine (ddI).¹⁰⁵ HU inhibits the cellular enzyme ribonucleotide reductase, thereby decreasing intracellular deoxyribonucleotide pools, which indirectly impedes viral RT activity.¹⁰⁶ As such, HU inhibits HIV-1 replication, notably in nonac­tivated CD4⁺ T cells, by indirectly blocking reverse transcriptase, which is dependent on intracellular deoxyribonucleotide triphosphates (dNTPs) as substrates.¹⁰⁶ Recently, it was demonstrated that mycophenolic acid, together with the RT inhibitor abacavir, may be similarly effective at blocking residual HIV-1 replication as ddI and hydroxyurea.¹⁰⁷ Hydroxyurea was used in several HAART regimens, especially combined with ddI, with which it has synergistic effects in inhibiting HIV-1 replication.¹⁰⁶ At least one patient had a dramatically low proviral reservoir in the peripheral blood when treated with a ddI and hydroxyurea-containing regimen and maintained undetectable plasma viral RNA after HAART was discontinued.⁵⁶ In addition, a small cohort of patients on ddI and hydroxyurea did not manifest viral rebound when the antiretroviral therapy was discontinued.¹⁰⁸ Unfortunately, another study showed that a quick and profound rebound of plasma HIV-1 developed after treatment with HAART, plus or minus hydroxyurea therapy during primary HIV-1 infection. One patient in this study, however, remained with fewer than 50 copies/ml of plasma HIV-1 RNA off HAART for 46 weeks.¹⁰⁹ However, this is an anecdotal case, and there remains a lack of compelling data from randomized, controlled studies.

Cytoreduction therapy may also be useful in removing persistently infected cells and available uninfected target cells. Whether cyclophosphamide as a low-level cytotoxic agent, or even HU at high levels, at which point it is cytotoxic,¹¹⁰ could be used in this manner will require further study. This approach may be based on the “predator and prey” relationship of HIV-1 with CD4⁺ T cells.¹¹¹ Targeting specific immunotoxins to deplete the HIV-1 proviral reservoir, after stimulation of viral transcription from the proviruses, and potentially ongoing viral replication should now also be readdressed in the era of HAART.¹¹²

As low levels of ongoing viral replication occur in many HIV-1-infected individuals on effective HAART, “intensification” therapeutics must be added to initial combination therapy to ablate this viral replication if eradication is a goal. Using approaches to purge only the latent viral reservoirs likely will not be successful. Additional studies on the penetration of different pharmacologic agents across blood-tissue barriers (e.g., blood-brain and blood-testes) will be of importance in attacking potential sanctuary sites that may contain both cryptically replicating as well as latently infected cells. Certainly, combinations of each of these approaches listed above may be required for elimination of the HIV-1 reservoirs in vivo. As such, one might now begin to think of the treatment of HIV-1 in somewhat of an “oncological” paradigm. This would include effective HAART as “induction” therapy and then additional approaches against HIV-1 latency, cryptic replication, and sanctuary sites for the removal of “residual disease.” This approach is not induction followed by “maintenance” therapy but induction followed by aggressive therapies to approach residual viral disease, with the induction HAART continued unaltered.

Immune-Based Approaches and Structured Treatment Interruptions (STIs)

As the potential for viral eradication remains an unproven concept and a difficult goal, studies aimed at disease remission have been developed. Gradual improvements in the immune system with effective HAART are tied to increased thymic and possibly extrathymic output of cells.¹¹⁴,¹¹⁵ Increased anti-HIV-1-specific immune responses have been detected in certain patients who were treated with virally suppressive HAART and then had it discontinued after various periods of time. There is a report of two patients who maintained undetectable levels of plasma viral RNA by clinical assays (fewer than 50 copies/ml).¹¹⁶ It is now being tested whether some patients may be placed into clinical remission and can be maintained off HAART for significant periods of time (see below). HIV-1-specific CD8⁺ cytotoxic T lymphocytes (CTLs), which usually become unde­tectable with fully suppressive HAART, increase when a significant level of viral RNA in plasma redevelops off HAART in patients who were virologically suppressed previously.¹¹⁷ In addition, HIV-1-specific CD4⁺ T lymphocyte proliferation, which is usually not found in patients first treated with suppressive HAART months to years after primary infection, remained detectable in infected individuals who were treated at the time of primary infection with fully suppressive HAART,¹¹⁸ although in one study, early but chronic infection treated with HAART demonstrated preserved anti-HIV-1 CD4⁺ T lymphocyte responses.¹¹⁹ These studies suggest that residual, antigen-specific CD8⁺ T lymphocytes may expand during cyclical periods of starting and stopping HAART.

Structured cyclic antiretroviral therapy interruptions are beginning to be studied in attempts to augment anti-HIV-1 immune responses. A few patients show a substantial decrease in plasma HIV-1 RNA set points after STIs. Although augmentation of select immune parameters may occur, there are many cases in these initial studies in which little or no virological effects were demon­strated.^120-125 Diverse antiretroviral immune responses seem to be well-preserved if suppressive HAART is initiated soon after primary HIV-1 infection in vivo.^118,126 A recent study showed exciting new data on remissions induced in a small group of patients treated with HAART during primary HIV-1 seroconversion, with subsequent treatment interruptions.¹²⁷ Although HIV-1 was not eradi­cated in these patients, quite low plasma viral RNA levels were induced and maintained off all antiretroviral therapy. Additional studies and long-term follow-up of these patients will be required to determine the durability of this effect and whether any patients initially treated with HAART during chronic HIV-1 infection will benefit from this approach. Unfortunately, STIs in chronically HIV-1-infected individuals treated after primary seroconversion, unlike STIs in patients treated during primary infection, have not been effective.¹²³ Selected therapeutic vaccines should also be used in future studies. Recent data in simian immunodeficiency virus (SIV)-infected macaques demonstrated viral control (i.e., with low plasma viral set points) when specific therapeutic vaccines were combined with STIs.¹²⁸

The potential use of selected immunotoxins, in synergy with augmentation of antiretroviral immune responses, toward maintaining HIV-1 remission was also proposed and awaits evaluation.¹¹² Exploring the hypothesis that the immune system may keep the virus in check (i.e., remission) and induce long-term nonprogression without leading to full viral eradication is an intriguing idea that clearly merits further clinical analyses. Converting HIV-1 infections into a manageable long-term disease may be based on the selection of less fit (i.e., lower replicative capacity) or less virulent (i.e., lower CD4⁺ T cell depletion) viral variants.

HAART has led to dramatic and positive changes in the treatment of HIV-1 infection. Nonetheless, in its present form, HAART is unlikely to lead to viral eradication in the majority of HIV-1-infected individuals within clinically reasonable time periods. Nonetheless, the progress obtained with therapy against HIV-1 within the last decade suggests cautious optimism regarding approaches for continued suppression of residual HIV-1 infection.

Conclusion

In summary, HAART had led to dramatic, positive changes in the clinical management of HIV-1 infection. However, it is not likely that these regimens will lead to elimination of virus in their present forms for the majority of HIV-1-infected individuals, at least within a clinically reasonable time frame. The presence of multiple persistent viral species sequestered in multiple cell types makes this a difficult task. Many questions remain open regarding the nature of HAART-persistent viruses and the cell and tissue types in which they may be sequestered.

The activation of specific subsets of patients’ cells in vitro with a variety of agents, apart from phytohemagglutinin (PHA) and IL-2 (used in many T cell activation and proliferation studies in cell cultures), is warranted. Compounds such as phorbol myristic acid (PMA) prostratin, IL-7, or IL-15 likely induce viral expression though a pathway distinct from PHA/IL-2. This is important, as latent virus may reside within multiple cell types, and each could have a heterogeneous response in terms of viral expression induced by individual activating agents. This possibility supports the use of a more diverse panel of stimulatory agents in vitro and, importantly, in in vivo studies. Certain mitogens, such as PHA, are restricted to use in vitro and are not viable candidates for in vivo clinical application.

Thus, further studies are necessary to try to identify not only which compounds would be able to induce viral expression from latent cellular reservoirs but also the molecular mechanisms involved in maintaining these viral reservoirs constant over time.

Finally, it is important to determine whether currently devised and clinically applied viral intensification and immune-stimulatory protocols are definitively aiding in the elimination of the latent viral pool or whether modifications to current protocols would enhance their efficacy. Such modifications could include use of additional stimulatory compounds or, alternatively, hybrid immu­notoxins that are specific recombinant proteins that exhibit preferential targeting and killing of HIV-1-infected cells.¹¹² Additional stimulatory molecules would broaden the spectrum of cell activation, which is linked to latent viral expression. These compounds, perhaps combined with novel immune-based therapies, could accelerate elimination of the virus so that complete withdrawal from HAART is realistically attainable for at least some HIV-1-infected individuals.

ACKNOWLEDGMENTS

The author wishes to thank Ms. Rita M. Victor and Ms. Brenda O. Gordon for excellent secretarial assistance.

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