Important note: Information in this article was accurate in May 2000. The state of the art may have changed since the publication date.Molecular aspects of HIV pathogenesis and transmission in vivo have been analyzed in detail for two decades, and understanding of these aspects of HIV infection has increased greatly over this period. As a result, many successes in anti-HIV therapy have been rationally designed, based on a clear understanding of the viral life cycle and HIV pathogenesis.1
In most infected individuals, HIV replicates at high levels throughout infection, including the clinical quiescent phase. Levels of this active viral replication directly correlate with disease progression and survival.2-4 Combination therapy for HIV--highly active antiretroviral therapy (HAART)--has dramatically altered viral replication in vivo, greatly decreasing HIV-related morbidity and mortality, at least in the developed world.5-7
A significant portion of therapeutically naive HIV-infected individuals treated with HAART attain undetectable plasma HIV RNA levels, as defined by a detection limit of 400 to 500 copies/mL, while somewhat fewer patients reach levels below 50 copies/mL.5,7 These low levels of plasma viremia now allow clearer investigations of classical questions in human retrovirology and, at the same time, generate new clinical problems to analyze (Table 1).
For example, with HAART's ability to reduce viral load (virion-associated RNA) to undetectable levels in blood plasma and genital fluids of many infected individuals, researchers can now formally analyze whether proviral-harboring genital tract cells can transmit HIV sexually, without cell-free virions. In addition, mechanisms of proviral persistence and cryptic viral replication can now be addressed without the "noise" of active virus-producing cells and high levels of cell-free virions.8
Persistently infected nonactivated CD4+ T lymphocytes have been demonstrated in the peripheral blood of HIV-infected individuals.9 Importantly, these provirus-harboring resting CD4+ lymphocytes have now been demonstrated in infected individuals treated with HAART and with undetectable viral RNA in blood plasma. Research has also shown that replication-competent virus can be recovered from these provirus-positive cells, after CD8+ T lymphocyte depletion in vitro.10-12 This latent reservoir is established soon after primary HIV seroconversion13 and can be activated by proinflammatory cytokines in vitro and potentially in vivo.14 A latent reservoir of low-level or nonreplicating HIV has also been described in HIV-infected children treated with suppressive HAART.15 In a short study by Poggi and colleagues,16 suppressive HAART begun before seroconversion was unable to halt the development of a replication-competent viral reservoir in CD4+ T lymphocytes. Another study suggested indirectly (by quantitating total HIV DNA and subtracting integrated HIV DNA) that some low-level viral replication may still take place in certain of these cells, because unintegrated viral DNA may be present.12 Earlier research showed that very low-level viral transcription may take place in lymph nodes of selected patients receiving effective HAART.17 Thus, it became clear that further studies were necessary to analyze the potential for cryptic viral replication in these cell populations.
Replication-competent viruses isolated from provirus-harboring T lymphocytes in patients with undetectable plasma viremia while receiving HAART were found to have few or no antiretroviral resistance mutations.10 Because resistance mutations in the reverse transcriptase (RT) and protease genes of HIV correlate with ongoing viral replication,18 this finding suggests that these viral strains may represent archival species from soon after seroconversion. These data also suggest that, although proviruses accumulate in CD4+ T lymphocytes in vivo (forming "viral graveyards"),19 replication-competent proviruses still exist in persistently infected cells. Persistence of this replication-competent virus may hinder attempts at eradication and reseed the body if HAART is discontinued in yet undetermined time periods after primary infection. Continued extensive analysis of in vivo decay times of these persistently infected cells is now critically important.
To investigate the impact of HAART on HIV replication in the male genital tract, our laboratories simultaneously collected peripheral blood and seminal fluids from seven HIV-infected men receiving prolonged triple antiretroviral therapy.8 All seven had repeatedly undetectable viral RNA in peripheral blood. Viral RNA in blood and seminal plasma on the days of sample collection was analyzed via quantitative RT-PCR, while cell-associated proviral HIV DNA from peripheral blood mononuclear cells (PBMCs) and seminal cells was evaluated by quantitative DNA-PCR.
Viral RNA levels in both blood plasma and seminal fluid of these patients were below 50 copies/mL. This finding suggests that HAART potently inhibited viral replication not only in the bloodstream of these individuals, but also in seminal fluids. Importantly, cell-associated viral DNA was detected in all patients' PBMC samples. Cell-associated proviral DNA was also detected in seminal cells from four patients.
Because levels of cell-free HIV in seminal plasma were very low or undetectable in these patients, we then examined whether HIV DNA-positive seminal cells could harbor replication-competent virus. Using a coculture system, we demonstrated HIV replication in seminal cells from two individuals. Since this report, we detected this seminal reservoir in additional patients (unpublished results).
Although HIV RNA was below detectable levels in seminal plasma of HIV-infected men in our study, reinfection of cells in the male genital tract tissues and fluids could still occur at very low levels (covert or cryptic replication), as has been demonstrated in lymphoid tissue.17 In this scenario, the life span of the cells harboring proviral DNA could still be relatively short. It remains to be clarified which specific cells in the genital tract of HIV-infected men receiving HAART harbor replication-competent HIV. Seeding of the male genitourinary tract may occur not only from the peripheral blood, but from reservoir sites in the genitourinary tract itself, including the prostate.20,21
We hypothesized that both latently infected cells containing nondefective but quiescent provirus, as well as low levels of viral replication, could account for the replication-competent virus in PBMCs and seminal cells of these infected individuals.8 At least in some cell types in these patients, viral replication could occur at such low levels that the virus would not be detectable in peripheral bodily fluids. But cryptic replication might produce enough virus to infect small numbers of cells in the surrounding microenvironment.
Two important articles clearly supported this hypothesis in at least some patients taking suppressive HAART. Using complementary techniques, Zhang and coworkers22 demonstrated modest but reproducible evolution of sequences in the envelope region of virus from two of eight patients taking suppressive HAART. Further studies demonstrated that, during what appears to be full suppression of HIV in the plasma by clinical assays, evolution of envelope sequences in cellular reservoirs indicates ongoing viral replication in most patients.23,24 As in previous studies,8,10 and in work by Furtado and colleagues,25 these archival viral strains did not carry antiretroviral resistance mutations, even in cryptically replicating strains.
The in vivo half-life of infectious residual HIV led Zhang and coworkers22 to estimate that viral eradication would require at least seven to 10 years of viral suppression with standard HAART, even in best-case scenarios. Interestingly, the data of Furtado and colleagues25 indicated a quasi-steady state of viral messenger RNA and integrated proviral DNA in their patients after 300 days of suppressive HAART.
The Furtado study25 used a sensitive measure of the "footprints" of cryptic replication, evaluating HIV long terminal repeat (LTR) DNA circles. These LTR circles are formed by self-ligation of proviral DNA, by cellular nuclear ligases, after DNA transport to the nucleus. Because of their potentially short in vivo half-lives, these viral DNA species signify at least low-level viral replication at some time in the recent past. This cryptic replication might "reset the virologic clock" by infecting previously uninfected cells in localized microenvironments.
Not all patients in either of these studies22,25 had changes in proviral envelope sequences or continuously present LTR circles suggesting ongoing viral replication. Thus, latently infected cells appear to exist in some patients, potentially intermixed with cells in which virus is cryptically replicating.
On the basis of these studies and other recent work involving patients analyzed during the HAART era, we can now rationally appraise the meaning of residual HIV disease after seemingly effective HAART. To further extend our understanding of residual cryptic HIV replication during suppressive HAART, we studied all patients initially referred to our clinic who were taking HAART and had fewer than 50 copies/mL of viral RNA in plasma. We used a supersensitive laboratory-based RT-PCR assay that can quantitate as few as 5 copies/mL and can detect but not quantitate viral RNA even below this level.26-28
The 22 patients in this study represented an immunologically diverse group, with CD4+ cell counts between 100 and 1270 cells/mm3, and 21 of 22 were treated with at least three antiretroviral agents.29 The stability of the patients' HAART regimens was an important criterion for inclusion in this cohort. We also evaluated virion RNA levels in genital secretions from each of these patients. As other investigators have demonstrated, RNA levels in genital secretions were usually lower than those found in peripheral blood plasma. Twelve patients had no detectable virion RNA in genital secretions, including both women analyzed in this study. Eight patients had detectable but unquantifiable viral RNA in seminal fluid. And two patients had quantifiable virion RNA in seminal fluid.
This study demonstrated that, in a cohort of HAART-treated patients with plasma viral RNA below 400 copies/mL for between five months and several years, and with fewer than 50 copies/mL in peripheral blood plasma at the time of these analyses, all subjects had low but detectable levels of HIV RNA in blood plasma.29 These results were surprising because they showed that viral expression could be demonstrated not only by viral replication in selected cell types in patients taking suppressive HAART, but also by actual virion production in blood plasma.
These findings correlated with data presented by several groups showing low levels of ongoing viral replication in selected patients by analysis of PBMCs and lymphoid tissue.17,25,30,31 Our studies expanded and extended these data. The sensitive techniques we used demonstrated that cryptic replication in certain cell types can be quantified by analysis of peripheral blood plasma. Importantly, this cryptic replication may result in infection not only of cells in the local microenvironment, but also of cells distant from the source of viral production. Thus, our results corroborate, on an intracellular level, the ongoing covert replication that occurs in most patients receiving suppressive HAART.32
It is still unclear which cell types produce these residual virions in patients taking a suppressive HAART regimen. Sequencing of viral RNA at these low levels is extraordinarily difficult, but this problem may be approachable with new technologies being developed. In the meantime, we cannot be certain whether this very low-level viral replication leading to virion particles in peripheral blood plasma occurs in peripheral blood lymphocytes, tissue-bound macrophages, lymph nodes, or other compartments that may be relative drug sanctuary regions, such as the central nervous system, retina, and testes.33 Further studies will be necessary to understand the in vivo molecular mechanisms behind "residual HIV disease" in patients receiving what is now clinically considered to be fully suppressive HAART.
Ongoing viral replication complicates analysis of the latent HIV reservoir. Recent studies suggested that some HAART-treated patients with fewer than 50 copies/mL of blood plasma RNA still have low-level bursts of viral replication leading to transient spikes above 50 copies/mL.34 In these patients, the latent replication-competent proviral reservoirs decayed much more slowly than in patients who consistently maintain plasma HIV RNA levels below 50 copies/mL. As such, there may be a viral reservoir caused by ongoing subclinical viral replication rather than slow decay of a truly latent proviral state. This reservoir appears to have a very long half-life and would require more than 60 years of suppressive therapy for eradication.35
The more recent study34 suggests at least some ground for optimism, since the latent reservoir's mean half-life of approximately six months in patients without spikes above 50 copies/mL may allow eradication in this select group of patients in substantially fewer than 60 years. Even in this select group of patients, however, other viral reservoirs in peripheral blood, lymphoid tissue, or other solid organs would lengthen the time to eradication. Preliminary results of another study demonstrate a biphasic decay of latent HIV in resting CD4+ T lymphocytes of individuals with primary HIV infection.36 This biphasic decrease may be due to differing decay rates of pre- versus postintegration latent viral DNA forms.
Treatment attempting to rid the body of HIV-infected cells, which can produce replication-competent virus in patients taking effective HAART, should be based on rational designs evolving from further understanding of the in vivo molecular pathogenesis of this human lentiviral disease. One could develop approaches to activate latently infected cells, as recently reported by Chun and colleagues with interleukin 2 (IL-2).37 Strategies could include infusion of IL-2, OKT3 antibodies, and even interferon gamma to activate monocyte/macrophages.
A preliminary study using anti-CD3 monoclonal antibodies plus IL-2 in an attempt to eradicate HIV in patients on suppressive HAART was unsuccessful.38 The eradication regimen increased viral replication and had serious side effects. Of note, though, the doses of IL-2 and OKT3 antibody were high in this pilot trial, and further studies will assess other doses of these agents. In addition, and most importantly, antiretroviral therapy was not intensified in this trial, so ongoing cryptic replication was probably not addressed in a significant manner.
A study in which patients taking suppressive HAART were also treated with intermittent IL-2 demonstrated that the latently infected pool of resting CD4+ T lymphocytes decreased and in some cases became undetectable.37 Nevertheless, when antiretroviral therapy was stopped in two patients in whom replication-competent virus could not be found in resting CD4+ T cells in vitro, viral replication resumed and plasma RNA levels went to high levels within a few weeks after halting therapy. Once again, these results suggest that merely adding stimulatory therapy with IL-2 may not have ablated ongoing viral replication.39 Another preliminary study evaluating lymphoid tissue also suggested that IL-2 combined with suppressive HAART has no clear effect on viral production or latent infection in vivo.40 In a recent study, Davey and colleagues interrupted HAART in a relatively large group of 18 patients with undetectable virus for over a year.41 Twelve of these patients were treated with IL-2 as well as HAART. All patients relapsed to relatively high levels of HIV RNA two to three weeks after stopping HAART. Thus, in this cohort and others,42 investigators clearly showed that neither long-term suppressive HAART alone nor HAART plus IL-2 led to remission of HIV infection or to eradication.
Another recent study showed that HIV-specific antigens can activate latently infected CD4+ T lymphocytes from patients taking suppressive HAART.43 This finding suggests that many latently infected resting CD4+ T cells actually have HIV antigen specificity. It may be possible to exploit this trait to reduce the reservoir of latently infected CD4+ T lymphocytes in vivo.
In addition, an in vitro study demonstrated that an anti-CD8 and anti-CD3 bispecific monoclonal antibody, when added to IL-2, is more efficient than IL-2 and anti-CD3 alone in activating latent HIV from infected individuals' PBMCs.44 Further studies such as this will be critical in determining the best approach toward activation and possible depletion of the latent HIV reservoir during suppressive HAART.
Much interest has developed around hydroxyurea, and further studies will continue to evaluate its potential role as an adjunct to antiretroviral therapy. Hydroxyurea has been used as a component of several HAART regimens, especially those containing didanosine (ddI), with which it has synergistic anti-HIV effects.45,46 This novel approach to antiretroviral therapy exploits hydroxyurea's selective inhibition of cellular ribonucleotide reductase. Inhibition of ribonucleotide reductase dramatically decreases intracellular deoxyribonucleoside triphosphate (dNTP) pools. As such, although hydroxyurea is not a primary antiretroviral agent, it inhibits HIV replication by indirectly blocking reverse transcriptase, which depends on intracellular dNTPs as substrates.47-49 Several clinical studies have demonstrated the in vitro and in vivo efficacy of hydroxyurea in inhibiting HIV replication when combined with ddI and other nucleoside reverse transcriptase inhibitors. Research also suggests that hydroxyurea's ability to limit the number of CD4+ T lymphocyte target cells may also contribute to this agent's in vivo activity when combined with antiretrovirals.50
Preliminary studies show that hydroxyurea-containing regimens profoundly inhibit viral replication, if started during primary HIV seroconversion (see below). Of note, at least one patient in a small cohort had a dramatically low proviral reservoir in the peripheral blood when treated with hydroxyurea, ddI, and a protease inhibitor and maintained undetectable viral loads after HAART was discontinued.51 Another group reported that two patients taking only ddI and hydroxyurea did not manifest viral rebounds when this treatment stopped.52 A third group, however, found that plasma HIV RNA rebounded quickly to high levels after withdrawal of treatment with HAART with or without hydroxyurea during primary HIV infection.53 Nevertheless, one patient in this study had fewer than 50 copies/mL of plasma HIV RNA 46 weeks after stopping HAART. Such cases suggest that early therapy may infrequently induce a "remission" of HIV replication.
It may also be worthwhile to study hydroxyurea for its potential effects on HIV reservoirs in patients who attain undetectable plasma RNA levels with HAART. Of note, hydroxyurea is a relatively small molecule that can cross the blood-brain barrier47-50 and so may also be able to cross the blood-testes barrier.
In addition, this agent can dramatically inhibit the processing of partial reverse transcripts into full-length reverse transcripts, a step necessary for viral integration into the host genome.47 If reverse transcription is normally delayed in certain genital tract cellular reservoirs, as it is in other cellular pools,54,55 hydroxyurea may further delay reverse transcription and decrease proviral integration in genital tract cells. This hypothesis suggests that hydroxyurea may be a prime candidate for decreasing or ablating the development of reservoirs of HIV provirus and replication-competent virus. Thus, it will be critical to assess the in vivo half-life of HIV reservoirs in patients taking hydroxyurea.
A recent study assessed hydroxyurea, ddI, and a protease inhibitor during acute HIV infection.56 This regimen led to undetectable plasma viremia (with clinical assays) and, importantly, decreased the latently infected CD4+ T lymphocyte reservoir in some of these patients. Other studies have shown, however, that HAART without hydroxyurea during HIV infection also allows a high proportion of patients to attain undetectable plasma viral RNA and may decrease the latent T-lymphocyte reservoir.57 A similar approach, using the nucleoside analog abacavir and the lymphocyte proliferation inhibitor mycophenolic acid, may also alter residual HIV replication.58
The potency of antiretroviral combination therapy may have some effect on HIV proviral DNA levels.59 Of note, though, much proviral DNA in vivo is defective and would not lead to active replication in any case. Therefore it may be quite stable, because of the long half-life of the T cells into which it is integrated.
In a recent study, suppressive HAART begun during primary HIV infection, although leading to clearance of virus detectable by a clinical assay (below 50 copies/mL), had little effect on proviral DNA in circulating CD4+ T lymphocytes.60 In a 48-week study, Ibanez and colleagues61 also showed that HAART did not lower levels of integrated proviral DNA from baseline. And another recent study demonstrated that patients treated with suppressive HAART during primary HIV infection still had ongoing cell-associated viral RNA in peripheral blood cells.62 Thus this study confirmed that potent and sustained antiretroviral therapy, even when started during primary infection, does not completely halt HIV replication in most cases.
Cytoreductive therapy may be useful in removing these persistent proviral- containing cells. Whether low-dose cyclophosphamide or even hydroxyurea can be used as a cytoreductive agent requires further study. Such a strategy would be based on the "predator-prey" relationship between HIV and CD4+ T lymphocytes, in which reducing the number of prey (activated T lymphocytes) limits the number of predators (HIV).63 Berger and coworkers64 have also suggested that targeting specific immunotoxins to deplete the HIV proviral reservoir--and potentially ongoing cryptic replication--should now be readdressed in the HAART era, rather than relying on data obtained before the advent of fully suppressive combination antiretroviral chemotherapy.
Intensification of the initial suppressive HAART regimen might also be considered as a way to ablate cryptic low-level replication, if viral eradication is the goal. Further studies of the penetration of different pharmacologic agents across blood-tissue barriers (eg, blood-brain and blood-testes barriers) will also be critically important in attacking potential sanctuary sites.
Certainly, combinations of each of the approaches discussed above may be required to eradicate HIV (Table 2). As such, one might begin to think of the treatment of HIV infection almost as an oncological treatment paradigm. Treatment would begin with effective HAART as induction therapy, then further approaches would address HIV latency, cryptic replication, and sanctuary sites to remove "residual disease."
In an interesting preliminary study,65 investigators detected increased anti-HIV-specific immune responses in certain patients who began HAART early in the course of infection. These six individuals discontinued or interrupted HAART after various points during the first year of treatment. Two patients seem to have maintained undetectable levels of plasma viral RNA (below 500 copies/mL) for more than 12 and more than 24 months after they stopped treatment. These individuals had broad and strong HIV-specific immune responses.
This observational study raised the question whether some patients may be placed into remission and kept off HAART for sustained periods. That possibility requires further analysis. Of note, though, levels of HIV-specific cytotoxic T lymphocytes (CTLs), which appear to become undetectable with fully suppressive HAART, do increase with substantial plasma viral load rebounds when HAART stops after suppression is attained.66 Data from Bruce Walker's laboratory also show that HIV-specific lymphocytes, which are not found when patients begin suppressive HAART months to years after seroconversion, remain detectable in infected individuals treated during primary HIV infection with fully suppressive HAART.67
These studies suggest that the immune system may actually be "trained" by cyclical periods of HAART begun during primary infection.65 Of interest, patients who attain undetectable levels of plasma viremia with HAART have weaker HIV-specific and nonspecific immune responses than individuals who maintain undetectable viremia without ever taking antiretroviral therapy.68
Several preliminary studies showed that cyclic structured treatment interruptions (STIs) can have a variety of effects. A few patients do have a decreased viral set point after STIs, but many patients in these early studies appear to derive little or no virologic benefit from this approach. Clearly, further studies will be necessary to determine whether STIs are effective in specific cohorts of HIV-infected individuals who attain undetectable plasma viremia with HAART.69-79 Studies have already clearly shown that diverse antiretroviral immune responses seem to be well preserved if suppressive HAART begins soon after HIV infection.80 Because the study by Ortiz and colleagues65 showed potent HIV-specific immune responses in patients with prolonged suppression of viral replication, the hypothesis that the immune system can keep the virus in check (that is, in remission) without leading to full viral eradication suggests the need for further investigation of this possibility.
In addition to ongoing studies of viral latency and replication during suppressive HAART, further analysis of CD4+ T-cell kinetics and trafficking will be important in understanding immune reconstitution and viral reservoirs.81,82 in vivo studies of changes in CD4+ T lymphocyte levels and kinetics will also be key in evaluating certain novel viral eradication therapies to be combined with HAART.83
HAART has led to dramatic positive changes in the treatment of HIV infection, at least in the developed world. However, in the vast majority of HIV-infected individuals, HAART in its present form will not lead to viral eradication.84 Nonetheless, the substantial progress in therapy against HIV infection over the last decade should encourage us to remain cautiously optimistic as we now face residual HIV disease in patients on HAART and continue the quest to eradicate HIV infection.
Roger J. Pomerantz, MD, is Professor of Medicine, Biochemistry and Molecular Pharmacology; Chief of Infectious Diseases; and Director of the Center for Human Virology at Thomas Jefferson University in Philadelphia.
I would like to thank Ms. Rita M. Victor and Ms. Brenda O. Gordon for excellent secretarial assistance. This work was supported in part by USPHS grants A146289 and AI38666, plus grants from Bristol-Myers Squibb and Ortho Biotech Pharmaceuticals.
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