HIV-1 replicates in most untreated infected individuals at high levels throughout the infection, including the clinical quiescent phase. Levels of this active viral replication directly correlate with disease progression and survival [1–4]. Combination therapeutics for HIV-1, or highly active antiretroviral therapy (HAART), has led recently to dramatic decreases in viral replication in vivo to below the clinical limits of detection (i.e. usually plasma HIV-1 RNA levels below 50–400 copies/ml, depending on the assay system utilized) and reductions in morbidity/mortality, at least in the developed world [5–7]. These therapeutic modalities have completely altered the epidemic in many regions. The era of HAART now allows both clearer investigations of classical questions in human retrovirology, as well as the generation of new clinical problems. Mechanisms of viral latency and hidden or ‘cryptic’ viral replication can also now be addressed without the ‘noise’ of active virally producing cells and high levels of cell-free virions , as virally suppressive HAART ‘unveils’ viral persistence. Furthermore, approaches towards possible viral eradication or at least long-term remission can be rationally studied. Nonetheless, it is critical to note that true viral eradication may be extremely difficult for many reasons (see below), including the finding that cells other than those in the immune system (e.g. kidney, heart, etc.) also appear to be infected at low levels with HIV-1 in vivo[1,9,10].
This review will discuss the mechanisms of HIV-1 persistence in vivo during the treatment of infected individuals with virally suppressive HAART. Both HIV-1 latency and low-level residual viral replication will be discussed in immune-based cells and other potential cellular reservoirs. These viral parameters will be correlated with the problems associated with maintaining long-term control of HIV-1 infections.
HIV-1 latency and persistence
Interest in retroviral latency or, more generally, persistence preceded the AIDS epidemic. Although retroviral latency was initially studied in avian retroviruses, the understanding of these processes in vivo has significantly increased in recent years, utilizing HIV-1 as a model . It is critical, at the outset, to delineate the concepts of cellular latency and persistent replication for retroviruses in vivo. Viral latency is a general property of many viruses, including herpesviruses. Retroviral latency is defined as integrated provirus with no active transcription. Low-level chronic yet productive viral expression best characterizes persistent or cryptic replication. In the case of HAART, virus replication from previously infected cells is not fully ablated. Viral persistence takes place after initially successful virally suppressive therapy or effective antiviral immune responses in the infected host. Such cryptic viral replication may also continue in immunologically privileged sites, such as the brain and testes. Transcriptionally active but non-productive viral infections (i.e. certain viral messenger RNA are expressed but not intact virions) may also occur (see below).
The HIV-1 replicative cycle contains many possible stages for latency and persistence via both pre- and post-integration into the human cell genome . Initially HIV-1 binds to cellular receptors and co-receptors (i.e. CD4 cell receptor and chemokine co-receptors, CCR5 and CXCR4), with subsequent viral core internalization and reverse transcription of the viral RNA template into a double-stranded DNA intermediate. The viral DNA is then integrated into the host cell genome as proviral DNA. Transcriptional activation of the integrated provirus is controlled by a complex series of interactions between HIV-1 regulatory proteins (e.g. Tat and Rev) and cellular transcription factors, controlled by the state of activation of the host cell. The activated provirus produces various viral mRNA species and, in some cases, new virions .
Many studies have demonstrated that ‘clinical’ HIV-1 latency, when defined as no viral expression in an entire untreated infected-individual, does not exist at the host level in any stage of disease [11–13]. In the great majority of HIV-1-infected individuals, some cultivable virus may be recovered at all stages of disease [11–13]. Nevertheless, data demonstrate that in the HIV-1-infected individual, some cells contain proviral DNA but express little or no viral RNA and produce few or no virions [11,14,15]. In addition to post-integration latency, various states of pre-integration HIV-1 latency have been described both in vitro and in vivo[16–20]. As such, latency at a cellular level exists in vivo, and the number of latently infected cells may vary based on the stage of disease. As HIV-1 infects, in vivo, both CD4 T cells, monocyte/macrophages, and other non-immune-based cells [10,21,22], the virus may maintain cellular latency by different mechanisms in differing cell types. This possibility presents important research opportunities in further elucidating the mechanisms of HIV-1 latency in vivo.
HIV-1 replicates at a rapid rate in infected individuals, with a virion half-life in plasma of hours . The vast majority of this viral replication (over 99%) occurs in activated and productively infected CD4 T cells in the peripheral blood and lymphoid tissue. Analysis of plasma viral decay characteristics in patients initially treated with potent combinations of antiretroviral drugs demonstrates a rapid first phase decay of productively infected cells, a second phase decay of long-lived cells (possibly from tissue-bound macrophages), and then a third phase decay of persistently infected cells (e.g. resting CD4 T cells)  (Fig. 1). Effective HAART blocks the virus from infecting healthy CD4 T cells, and the initial drop in HIV-1 levels in the peripheral blood reflects the life-spans of cells that were infected before treatment was initiated. On the basis of these complex viral and cellular kinetics with slow decay of viral infected cells in several phases, most HIV-1-infected individuals would require complete suppression of viral replication for years if one is even going to consider viral eradication. However, this goal is hindered by several factors, including less than optimal treatment, HIV-1 infection in immunologically privileged sites, and partial drug sanctuary compartments in vivo (see below). This virus–cell kinetics model (Fig. 1) has been challenged in some of its aspects on empirical grounds, in the context of ‘spikes’ or ‘blips’ of plasma viremia occurring in certain patients on virally suppressive HAART, and potential on-going, de novo cellular infections, secondary to local bursts of viral replication .
Residual HIV-1 provirus in vivo during highly active antiretroviral therapy
Persistently infected, non-activated CD4 T cells have been demonstrated in the peripheral blood of HIV-1-infected individuals after treatment effectively suppresses most productive viral infection . Replication-competent viruses can be recovered from these proviral-positive cells after CD8 T cell depletion in vitro[26–28]. The vast majority of these viruses are CCR5 tropic . In addition, this cell reservoir is established soon after primary HIV-1 infection , and can be activated by proinflammatory cytokines in vitro, and potentially in vivo. Suppressive HAART initiated before primary HIV-1 seroconversion and during perinatal HIV-1 infection was also shown to be unable to halt the development of this replication-competent virus reservoir in CD4 T cells [30,32,33].
In a noteworthy recent study , latent replication-competent HIV-1 provirus was demonstrated in mainly resting memory (CD45RO) CD4 T cells, but low levels of virus were also found in resting naive (CD45RA) CD4 T cells in patients on virally suppressive HAART. It was suggested that naive CD4 T cells are rarely directly infected but that the provirally harboring naive CD4 T cells are generated via reversion from a memory phenotype . In addition, the direct infection of thymocytes may also lead to the infection of naive T cells .
Most viruses isolated from resting CD4 T cells from the peripheral blood of patients with undetectable levels of viral RNA in plasma have few mutations that confer antiretroviral drug resistance . As resistance mutations in the reverse transcriptase and protease genes of HIV-1 are correlated with on-going viral replication despite treatment , this finding suggests that these viral strains may represent ‘archival’ species from a time before treatment. Of note is the fact that a recent study has shown that in some patients on virally suppressive HAART, with transient spikes of virus above 50 copies/ml in plasma, resistance mutations may develop . Data from the above studies demonstrated that although defective proviruses accumulate in CD4 T cells in vivo (i.e. ‘viral graveyard sequences') , replication-competent provirus still exists in resting CD4 T cells, which may hinder attempts to reduce the viral reservoir and re-seed the body with virus if HAART is discontinued. These possibilities, therefore, confound our attempts at pharmacologically mediated viral eradication. In addition, a recent study  demonstrated replication-competent HIV-1 in the peripheral blood monocytes of patients on virally suppressive HAART.
Tissues that maintain blood–tissue barriers, secondary to microvascular endothelial cell fight junctions, may limit the penetration of certain antiretroviral agents and act as partial ‘drug sanctuaries'. These compartments would potentially include the central nervous system, the retina, and the testes [1,8,39]. Although a recent article suggests this may not be likely in most patients (39a). A plasma membrane localized drug transporter, the P-glycoprotein, has been shown to decrease the penetration of protease inhibitors across certain blood–tissue barriers. As such, substances that block this drug transporter will increase protease inhibitor concentrations across the blood–tissue barriers. This finding may represent a new pharmacological approach to target relative viral sanctuary sites with higher levels of antiretroviral agents . Nevertheless, most analyses do show the parallel decay of HIV-1 in tissues, compared with peripheral blood, in patients on virally suppressive HAART . In addition, free-drug levels, rather than protein-bound drug concentrations, have rarely been analysed in body sites outside the peripheral blood.
Active replication of HIV-1 during highly active antiretroviral therapy
Persistently infected CD4 T cells containing non-defective but quiescent HIV-1 proviral DNA and low levels of viral replication could account for the replication-competent virus within the peripheral blood CD4 T cells and seminal cells of infected individuals on HAART . Viral replication could occur at such low levels that the virus would not be detectable in peripheral body fluids using standard clinical assays. Low-level productively infected cells might infect small numbers of target cells in the surrounding cellular microenvironment. One study  demonstrated that, in a minority of patients on suppressive HAART, a modest evolution of viral envelope sequences occurred over time. Other studies [43,44] demonstrated that during what appears to be full suppression of HIV-1 in the plasma, as determined by clinical RNA assays, there is ongoing viral replication in a majority of patients in some cohorts, as shown by the evolution of viral sequences in cellular reservoirs. These ‘archival’ viruses did not demonstrate antiretroviral resistance mutations, even in those strains that could undergo low-level replication. Nonetheless, some selection bias of the viral genetic variants may confound analyses in such studies.
In studies by Furtado et al. recently confirmed by another group , sensitive measures of the ‘footprints’ of persistent viral replication were used to evaluate HIV-1 mRNA species and HIV-1 long terminal repeat (LTR) DNA circles, which are formed by self-ligation of proviral DNA by cellular nuclear ligases after transport of the viral pre-integration complex to the nucleus. LTR-DNA circles were demonstrated in the CD4 T cells of most patients on suppressive HAART. The LTR circles, along with quasi-steady-state levels of HIV-1 mRNA, demonstrate the existence of low-level viral replication at some time in the recent past. Although a short in-vivo half-life for HIV-1 2-LTR DNA circles has been suggested , this is now controversial and further studies are necessary to confirm this finding.
Our laboratories have recently demonstrated that levels of in-vivo cryptic HIV-1 replication differ in specific patients on virally suppressive HAART, and can be used to stratify these groups of patients with residual retroviral disease .
In one study , a further complexity towards characterizing HIV-1 persistence during HAART was also demonstrated, as certain patients had cells with multiply spliced RNA out of proportion to unspliced viral RNA, in agreement with in-vitro models [48–50]. As multiply spliced viral RNA encodes regulatory proteins, whereas unspliced RNA encodes structural proteins, this pattern suggests transcriptionally active but non-productive infection. These patterns of viral RNA species expression may be based on the state of host cell activation. Several forms of cryptic replication might thus ‘reset the virological clock’ by infecting previously uninfected cells in localized microenvironments.
Recent data have demonstrated that certain unstimulated, HLA-DR-negative CD4 T cells in lymphoid tissue sampled from HIV-1-infected individuals treated with virally suppressive HAART were positive for low levels of viral RNA . HIV-1 replication was demonstrated previously to occur in activated memory CD4 T cells in both peripheral blood and lymphoid tissues [1,41]. These data suggest that there may actually be a spectrum of cell-types in a relatively inactive state that can still express low levels of viral RNA, a finding supported by others .
Most [53,54], but possibly not all , patients rebound rapidly with high levels of plasma viral RNA when standard suppressive HAART is discontinued, even when less than 50 copies/ml of plasma viral RNA has been demonstrated for significant time-periods. This observation suggests not only the presence of persistently infected cells but also on-going viral replication. One study  showed that rebounding virus in certain patients, after the discontinuation of HAART, did not appear to be from an outgrowth of latent provirus in resting peripheral blood CD4 T cells, but the cellular reservoir that produces the rebound virus remained unclear. Nevertheless, another recent study  demonstrated that rebound virus in some cases can arise from the latent virus in resting CD4 T cells.
Using a laboratory-based reverse transcriptase–polymerase chain reaction assay that can quantify at least 5 copies/ml of plasma viral RNA , low but detectable levels of virus were demonstrated in all HIV-1-infected patients studied with clinically undetectable levels of plasma HIV-1 RNA (i.e. less than 50 copies/ml) . Of importance was the fact that this ongoing viral replication may infect cells in local sites, as well as cells at a distance within the body. Of note, however, the quantities of defective virions  in residual plasma HIV-1 are still unknown and remain important for understanding the possible viral spread within an infected host.
The viral decay characteristics in cells are different from viral decay characteristics in plasma . In peripheral blood mononuclear cells the first phase represents the decay of productively infected and long-lived cells, whereas the second phase is the decay of cell-associated viral DNA and mRNA. Viral reservoir decay characteristics in one analysis  have suggested that 60 years will be necessary for patients treated with suppressive HAART to continue therapy for potential viral eradication. Unfortunately, even this estimate may not represent a best-case scenario for patients, as this analysis did not fully take into account low-level viral replication in tissues apart from the lymphoid organs.
Some patients with less than 50 copies/ml of blood plasma viral RNA on HAART still have low-level bursts of viral replication leading to more than 50 copies/ml in transient ‘spikes’, which carry risks of viral resistance evolution . The mean decay half-life of latent replication-competent proviral reservoirs is longer in patients with transient plasma viral RNA spikes, compared with those patients who consistently maintain plasma HIV-1 RNA levels below 50 copies/ml . A recent small study  suggested that low-level spikes of plasma HIV-1 RNA in patients on previously suppressive HAART did not appear to associate with higher rates of viral rebound, at least in the short term. Other preliminary studies [63,64], however, showed lower CD4 T cell counts in patients with plasma HIV-1 RNA spikes. It will be important to determine whether these viral spikes are caused by insufficient plasma drug levels or depressed drug penetration into unique reservoir sites, or just continued HIV-1 replication in the lymphoidal tissues, as well as the peripheral blood cells.
The findings that 60 years of suppressive HAART would be necessary possibly to allow latent reservoir eradication were suggested to be caused more by the slow decay of a truly latent proviral state rather than by ongoing viral replication . The other study mentioned above  might lead to some optimism, as a mean half-life of approximately 6 months of the latent viral reservoir in patients who have no spikes above 50 copies/ml of plasma viral RNA on HAART may allow viral eradication in this very select group of patients in a shorter time-period. Nonetheless, once again this possibility assumes that even in these patients there exists no other viral reservoir(s) in peripheral blood, lymphoid tissue, or other solid organs , and that sub-clinical viral replication did not occur.
Follicular dendritic cells
Mathematical modeling to evaluate the dissociation of HIV-1 virions from follicular dendritic cells (FDC) in lymph nodes during suppressive HAART suggests that HIV-1 virions bound to FDC decrease after HAART, and that this process may take up to 30 months . A recent model extends these data and suggests that a biphasic decrease in virions occurs, with the second phase being quite long, thus it may take up to several years for complete ablation of FDC-bound virions on suppressive HAART. This model is predicated upon the complete suppression of viral replication [65,66]. Virions bound to FDC could thus also be important in negatively affecting attempts at HIV-1 eradication. This virion binding may be based on the recently described DC-SIGN receptor on FDC . See Table 1 and Fig. 2 for virus–cell interactions during virally suppressive HAART in vivo.
Treatment of residual HIV-1 disease
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 (Table 2). On the basis of our understanding of molecular lentiviral pathogenesis, approaches can be developed to activate persistently infected cells, which might lead to virus-induced cell death and purging of the viral reservoir. In theory, activation approaches for eliminating persistently infected cells would not lead to increases in newly infected cells, virus spread would be prevented by HAART. This approach was attempted recently utilizing solely IL-2 , but it might also include infusions of anti-CD3 antibodies and even IFN-γ for activating monocyte/macrophage scavenger cells. An initial study  using anti-CD3 monoclonal antibodies plus IL-2 was unsuccessful in trying to eliminate HIV-1 in patients on suppressive HAART. Patients had increased viral replication and experienced serious side-effects from the high doses of drugs. No intensification therapy (i.e. additional antiretroviral drugs) was added in this clinical trial, in attempts to halt low-level viral replication . Therefore, persistently infected cells were stimulated, but the on-going cryptic viral replication was probably not affected in a substantial manner.
Some patients on suppressive HAART treated with intermittent IL-2 had decreased or undetectable levels of resting CD4 T cells containing replication-competent virus . 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 . Another larger group of 18 patients with undetectable levels of virus for more than one year had therapy interrupted . All patients relapsed to relatively high levels of plasma HIV-1 RNA in 2–3 weeks after stopping HAART. Neither relatively long-term suppressive HAART alone nor this treatment combined with IL-2 led to the elimination of HIV-1 infection. Such studies suggested that ‘rebound’ viremia after stopping HAART with IL-2 may be produced from yet unidentified reservoir sites.
Hydroxyurea has been used in several HAART regimens, especially combined with didanosine, with which it has synergistic effects in inhibiting HIV-1 replication . Hydroxyurea selectively inhibits cellular ribonucleotide reductase, a scavenger for tyrosyl-free radicals, which is essential for enzyme activity. This effect leads to a dramatic decrease in intracellular deoxyribonucleoside triphosphate (dNTP) pools. As such, hydroxyurea inhibits HIV-1 replication, notably in non-activated CD4 T cells, by indirectly blocking reverse transcriptase, which is dependent on intracellular dNTP as substrates . Mycophenolic acid, by affecting dNTP concentrations combined with the reverse transcriptase inhibitor, abacavir, may also have an effect on residual HIV-1 replication similar to didanosine and hydroxyurea [73,74].
At least one patient had a dramatically low proviral reservoir in the peripheral blood, when treated with a didanosine and hydroxyurea-containing regimen, and maintained undetectable plasma viral RNA after HAART was discontinued . In addition, a small cohort of patients on didanosine and hydroxyurea did not manifest viral rebound when the antiretroviral therapy was discontinued . 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 the study remained with less than 50 copies/ml of plasma HIV-1 RNA off HAART for 46 weeks . However, this was an anecdotal case, and there remained a lack of compelling data from randomized, controlled studies, and certain safety issues exist, regarding the use of hydroxyurea in inducing ‘remissions’ in HIV-1 infection. Cytoreduction therapy may also be useful in removing both persistently infected cells and available uninfected target cells. Whether cyclophosphamide, as a low-level cytotoxic agent, or even hydroxyurea at high levels at which it is cytotoxic , could be utilized 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 [78,79]. 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 re-addressed in the era of HAART .
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. Utilizing approaches to purge only the latent viral reservoirs will probably not be successful. In addition, further studies on the penetration of different pharmacological agents across blood–tissue barriers (e.g. blood–brain and blood–testes) will be of importance in attacking potential sanctuary sites, which may contain both cryptically replicating as well as latently infected cells (Fig. 2). Certainly, combinations of each of these approaches listed above may also be required for the 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 further 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. In any case, HAART alone decreases mortality and morbidity in the vast majority of patients maintaining a low viral load, despite residual HIV-1 infection, even in subjects with virological ‘failure’ and drug resistance .
Immune-based approaches and structured treatment interruptions
As the potential for viral eradication remains an unproved 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 extra-thymic output of cells [82,83]. Increased anti-HIV-1-specific immune responses were 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 (below 50 copies/ml) . 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 (CTL), which usually become undetectable with fully suppressive HAART, do increase when a significant level of viral RNA in plasma re-develops again off HAART, in patients who have been virologically suppressed previously . 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 . In one study, known early but chronic infection treated with HAART did demonstrate preserved anti-HIV-1 CD4 T lymphocyte responses . These studies suggest that residual, antigen-specific CD8 T lymphocytes may expand during cyclical periods of starting and stopping HAART. Nevertheless, these findings require further controlled studies to validate the clinical relevance of the alterations of specific anti-HIV-1 immunological parameters.
Structured cyclic antiretroviral therapy interruptions have begun to be studied in attempts to augment anti-HIV-1 immune responses. A few patients do show a substantial decrease in plasma HIV-1 RNA ‘set-points’ after structured therapy interruptions. Although the augmentation of select immune parameters may occur, there are many cases in these initial studies in which little or no virological effects were demonstrated [88–104]. Diverse antiretroviral immune responses do seem to be well preserved if suppressive HAART is initiated soon after primary HIV-1 infection in vivo[86,105]. 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. Although HIV-1 was not eradicated in these patients, low plasma viral RNA levels resulted and were maintained off all antiretroviral therapy. Further 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, structured treatment interruptions in chronically HIV-1-infected individuals treated after primary seroconversion, unlike structured treatment interruptions in patients treated during primary infection, have not been very effective . Selected therapeutic immunization should also be utilized in future studies. Recent data in SIV-infected macaques demonstrated viral control (i.e. with low plasma viral ‘set-points'), when specific immunization with viral proteins was combined with structured treatment interruptions .
The potential use of selected immunotoxins, in synergy with the augmentation of antiretroviral immune responses, towards maintaining HIV-1 ‘remission', has also been proposed and awaits evaluation . Exploring the hypothesis that the immune system may keep the virus ‘in check’ (i.e. remission) and induce long-term non-progression 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 also possibly be based on 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 virus elimination in the majority of HIV-1-infected individuals within clinically reasonable time periods. Nonetheless, the progress obtained with therapy against HIV-1 within the past decade suggests cautious optimism regarding approaches for the continued suppression of residual HIV-1 infection.
The author wishes to thank Drs Hui Zhang, Robert Siliciano, Ian Frank, and Edward Berger for helpful discussions, and Ms Rita M. Victor and Ms Brenda O. Gordon for excellent secretarial assistance. This work was supported, in part, by USPHS grant AI4628940 to R.J.P.
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