Residual HIV-1 infection during antiretroviral therapy: the challenge of viral persistence (2001)

Introduction 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 [8], 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 [1]. 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 [1]. 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 [1]. 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 [23]. 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) [23] (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 [24].Fig. 1.: The in-vivo dynamics and cellular reservoirs for HIV-1. This figure illustrates the different cell types infected with HIV-i in vivo and the possible sites for long-lived infected cell populations and latently infected cells (adapted with permission from R. Pomerantz, HIV-1 in semen and other reservoirs. In: HIV: advances in research and therapy, Vol. 9. Darien, CT: Cliggott Publishing Co.; 1999). Red, Viral genomic RNA; Green, integrated or unintegrated viral DNA; Blue, host cell chromosome. Upper left, productively infected cell; Upper right, pre-integration latently infected; Lower left, integrated latently infected; Lower middle, long-lived cell populations; Lower right, cells with defective proviruses.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 [25]. 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 [29]. In addition, this cell reservoir is established soon after primary HIV-1 infection [30], and can be activated by proinflammatory cytokines in vitro, and potentially in vivo[31]. 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 [29], 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 [29]. In addition, the direct infection of thymocytes may also lead to the infection of naive T cells [34]. 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 [26]. As resistance mutations in the reverse transcriptase and protease genes of HIV-1 are correlated with on-going viral replication despite treatment [35], 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 [36]. Data from the above studies demonstrated that although defective proviruses accumulate in CD4 T cells in vivo (i.e. ‘viral graveyard sequences') [37], 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 [38] 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 [40]. Nevertheless, most analyses do show the parallel decay of HIV-1 in tissues, compared with peripheral blood, in patients on virally suppressive HAART [41]. 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 Cell-associated virus 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 [8]. 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 [42] 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.[45] recently confirmed by another group [46], 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 [46], 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 [47]. In one study [45], 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 [51]. 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 [52]. Most [53,54], but possibly not all [55], 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 [56] 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 [57] demonstrated that rebound virus in some cases can arise from the latent virus in resting CD4 T cells. Cell-free virus Using a laboratory-based reverse transcriptase–polymerase chain reaction assay that can quantify at least 5 copies/ml of plasma viral RNA [58], 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) [59]. 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 [37] 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 [2]. 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 [60] 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’[61], which carry risks of viral resistance evolution [36]. 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 [61]. A recent small study [62] 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 [60]. The other study mentioned above [61] 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 [10], 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 [41]. 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 [67]. See Table 1 and Fig. 2 for virus–cell interactions during virally suppressive HAART in vivo.Fig. 2.: Residual HIV-1 infection during suppressive highly active antiretroviral therapy. This schematic illustrates known and potential sites for HIV-1 residual disease during virally suppressive highly active antiretroviral therapy (HAART). These include low-level productively infected CD4 T cells, transcriptionally active but non-productive CD4 T cells expressing mainly multiply spliced viral RNA, and latently infected resting CD4 T cells. Other potential sites include follicular dendritic cells to which virions are bound and may exist in this form for significant time periods in vivo. Potential sanctuaries for both cryptically replicating and latently infected cells are demonstrated behind various blood–tissue barriers. Macrophages that may be latently infected or cryptically replicating are also illustrated, and other potential diverse cell types for HIV-1 residual disease are listed on the left of this figure. I, Cells infected with HIV-1.Table 1: Factors in the persistence of HIV-1 infection. Treatment of residual HIV-1 disease Stimulatory therapy The treatment options for to the body of cells infected with HIV-1, which can produce replication-competent virus in patients on effective HAART, are now to be On the basis of our understanding of approaches can be developed to persistently infected cells, which might lead to cell and of the viral In activation approaches for persistently infected cells would not lead to in infected cells, virus spread would be by HAART. This approach was recently utilizing but it might also include of and even for cells. initial study using was in to HIV-1 in patients on suppressive HAART. had increased viral replication and from the high of therapy (i.e. antiretroviral was in this clinical in attempts to halt low-level viral replication [59]. persistently infected cells were but the on-going cryptic viral replication was not in a Potential therapeutic options for HIV-1-infected individuals or virally highly active antiretroviral therapy. Some patients on suppressive HAART treated with had or undetectable levels of resting CD4 T cells containing replication-competent virus treatment was in patients in replication-competent virus could not be found in resting CD4 T cells, virus was isolated in vitro and high levels of viral RNA in plasma were within of therapy group of patients with undetectable levels of virus for more than one had therapy patients to relatively high levels of plasma HIV-1 RNA in after HAART. relatively long-term suppressive HAART this treatment with led to the of HIV-1 infection. Such studies suggested that viremia after HAART with may be from yet reservoir therapy has been used in several HAART with with which it has in HIV-1 replication cellular a for which is for This to a dramatic decrease in As such, HIV-1 in non-activated CD4 T cells, by reverse which is on as by affecting concentrations with the reverse transcriptase may also have an on residual HIV-1 replication to and least one had a low proviral reservoir in the peripheral blood, when treated with a and and undetectable plasma viral RNA after HAART was In addition, a small of patients on and did not viral rebound when the antiretroviral therapy was Unfortunately, another study showed that a and rebound of plasma HIV-1 developed after treatment with HAART, or therapy during primary HIV-1 infection. One in the study remained with less than 50 copies/ml of plasma HIV-1 RNA HAART for However, this was an and there remained a of data from controlled and certain the of in in HIV-1 infection. therapy may also be in both persistently infected cells and uninfected target cells. as a low-level or even at high levels at which it is could be in this will require further This approach may be based on the and of HIV-1 with CD4 T cells specific to the HIV-1 proviral after of viral transcription from the and potentially ongoing viral replication now also be in the era of HAART As low levels of ongoing viral replication occur in many HIV-1-infected individuals on effective HAART, therapeutics be to initial therapy to this viral replication if eradication is a approaches to only the latent viral reservoirs will not be In addition, further studies on the penetration of different pharmacological agents across blood–tissue (e.g. and will be of importance in potential sanctuary sites, which may contain both cryptically replicating as well as latently infected cells (Fig. combinations of of these approaches listed above may also be for the of the HIV-1 reservoirs in vivo. As such, one might now to of the treatment of HIV-1 in of an This would include effective HAART as therapy and then further approaches HIV-1 latency, cryptic and sanctuary sites for the of This approach is not by therapy, but by to approach residual viral with the HAART continued In any HAART decreases and in the vast majority of patients maintaining a low viral despite residual HIV-1 infection, even in with virological and drug resistance approaches and treatment As the potential for viral eradication an and a difficult studies at disease remission have been in the immune system with effective HAART are to increased and possibly of cells immune responses were in certain patients who were treated with virally suppressive HAART and then had it after various periods of time. is a of patients who undetectable levels of plasma viral RNA by clinical 50 copies/ml) It is now being whether some patients may be into clinical and can be HAART for significant periods of time (see below). CD8 T which usually undetectable with fully suppressive HAART, do increase when a significant level of viral RNA in plasma again HAART, in patients who have been previously In addition, CD4 T 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 known but chronic infection treated with HAART did demonstrate CD4 T responses These studies suggest that CD8 T may during periods of and HAART. Nevertheless, these findings require further controlled studies to the clinical of the of specific antiretroviral therapy have to be studied in attempts to immune A few patients do show a decrease in plasma HIV-1 RNA after therapy Although the of select immune parameters may there are many cases in these initial studies in which little or no virological were demonstrated antiretroviral immune responses do to be well if suppressive HAART is initiated soon after primary HIV-1 infection in A recent study showed new data on in a small group of patients treated with HAART during primary HIV-1 with subsequent treatment Although HIV-1 was not in these patients, low plasma viral RNA levels and were all antiretroviral therapy. studies and long-term of these patients will be to determine the of this and whether any patients initially treated with HAART during chronic HIV-1 infection will from this Unfortunately, treatment in HIV-1-infected individuals treated after primary treatment in patients treated during primary infection, have not been very effective therapeutic also be in studies. Recent data in demonstrated viral control (i.e. with low plasma viral when specific with viral proteins was with treatment The potential of in with the of antiretroviral immune towards maintaining HIV-1 has also been and the that the immune system may the virus (i.e. and long-term without leading to full viral eradication is an that further clinical HIV-1 infections into a long-term disease may also possibly be based on selection of less (i.e. lower replicative or less (i.e. lower CD4 T cell viral HAART has led to dramatic and positive in the treatment of HIV-1 infection. Nonetheless, in its HAART is to lead to virus in the majority of HIV-1-infected individuals within clinically time Nonetheless, the with therapy HIV-1 within the suggests approaches for the continued suppression of residual HIV-1 infection. The to and for and and for This was in by to