Several mechanisms may contribute to the persistence of viremia during effective HAART. These include the inability of HAART to completely suppress virus replication because of inadequate potency (14
), intermittent nonadherence resulting in suboptimal drug concentrations (30
), and the emergence of drug-resistant variants (19
). Ongoing cycles of replication in the setting of HAART could lead to the accumulation of drug resistance mutations and treatment failure. Another possible explanation for ongoing low-level viremia is the continued production of HIV-1 by infected cells harbored in viral reservoirs or drug sanctuary sites (1
). One such reservoir that is established during acute infection is a small pool of latently infected resting memory CD4+
T cells (5
). This latent reservoir has been shown to retain HIV-1 in a replication-competent form despite many years of suppression of viremia to <50 copies/ml (6
Our study of 15 children with durable suppression of HIV-1 replication treated with HAART for up to 6 years demonstrates that HIV-1 viremia persists at plasma virus levels below 50 copies/ml in most if not all infected children on HAART. Viremia was even detected in a unique subset of children who started HAART in early infancy, some of whom reverted to HIV-1 seronegativity. We were able not only to detect viremia but also to clone and characterize the protease gene from the rare plasma virions that constitute viremia at this level. Amplification from low numbers of template molecules raises concerns about contamination, but phylogenetic analysis confirmed the patient-specific nature of the plasma viral sequences obtained. Most importantly, despite the finding of continued virus production in the setting of PI-based HAART regimens, HIV-1 viremia in children achieving durable suppression with their first PI-HAART regimen consisted primarily of HIV-1 variants that were wild type in protease. For the six subjects monitored longitudinally (C2, C7, C8, C10, C22, and C40), the median duration of continuous exposure to PIs was 3.2 years (range, 0.59 to 5.12). The median duration of follow-up was 21 months (range, 5 to 37 months). Despite the length of the observation period, accumulation of mutations in protease was not observed. These findings suggest that persistent low-level viremia is characteristic of HIV-1 infection during effective HAART and is comprised largely of PI-susceptible HIV-1 variants rather than protease variants with some degree of PI resistance.
Two potential processes might be involved in the stable persistence of HIV-1 viremia in the setting of potent antiretroviral therapy. One possibility is that viremia at plasma RNA levels of <50 copies/ml represents a new steady state, reflecting ongoing cycles of HIV-1 replication. Under these conditions, HIV-1 replication might be continuous but might occur only at low levels because of efficacy of the HAART regimen. Indeed, studies in HIV-1-infected adults have shown that the decay kinetics of plasma HIV-1 RNA during initial HAART can be accelerated when four- or five-drug HAART regimens are used (14
). This finding suggests that during standard HAART with three antiretroviral agents, newly infected cells are generated, albeit at a low level. If the stable persistence of HIV-1 viremia observed during HAART is in fact due to continuous cycles of HIV-1 replication, one might expect the genetic composition to show progressive divergence and the eventual emergence of drug-resistant variants (13
). However, neither drug-resistant HIV-1 nor viral divergence was observed in the children who were fully compliant, as evidenced by durable suppression with their first PI regimen. It remains possible that the pace of evolution of drug resistance in protease is too slow for detection over the treatment period (36
). The pol
gene is more stable than other regions (e.g., env
) of the HIV-1 genome; therefore, it is possible that analysis of changes in env
sequences over time might provide some evidence for evolution (16
). Nevertheless, the evolutionary changes that are likely to be of clinical significance in these patients are the changes affecting drug susceptibility, and these were not apparent in our study.
The persistence of low-level viremia in children on HAART can also be understood in the context of the latent reservoir for HIV-1. Although resting CD4+
T cells harboring replication-competent HIV-1 are present at a low frequency (1 per million resting CD4+
T cells), they can be readily detected using cellular activation methods in all infected individuals (6
). It is therefore plausible that resting CD4+
T cells harboring latent replication-competent HIV-1 are continuously activated for virus production during HAART. In our phylogenetic analysis, we found extensive commingling of protease sequences from plasma virus and replication-competent HIV-1 recovered from resting CD4+
T cells during HAART. Several genetic studies in HIV-1-infected adults have implicated viral reservoirs, including the resting CD4+
T-cell reservoir, as sources of rebound viremia following treatment discontinuation (4
). In patients failing antiretroviral therapy with multidrug-resistant HIV-1, wild-type HIV-1 becomes predominant when the drugs are discontinued (7
). The only documented site where archival, wild-type, replication-competent HIV-1 can coexist for long periods of time with drug-resistant variants generated during treatment failure is the latent reservoir in resting CD4+
T cells (38
). Therefore, the reemergence of wild-type HIV-1 in the plasma of patients who clearly have had viral divergence with progressive high-level drug-resistant virus provides further evidence for the active contribution of latent viral reservoirs to plasma virus. The continued production of drug-sensitive HIV-1 that we observed in children on suppressive HAART can be explained by the release of virus from stable reservoirs.
The failure to detect PI-resistant variants in most patients was not due to any inherent bias in the assay. Possible early PI-resistant variants were detected in two children (C7 and C40) who had no history of failure on a PI regimen, one with suboptimal adherence and the other in the setting of fully suppressive HAART. In patient C7, a V82I substitution was present in all clones amplified during a blip to 148 copies/ml following recent nonadherence. This substitution, while not typically observed with nelfinavir failure, could represent a preexisting polymorphism or an uncommon early resistance mutation (23
). At 3 months later, the viral load was <50 copies/ml and we were unable to detect persistent low-level viremia with our assay. In patient C40, changes at two sites, a N88S mutation at the first analysis and a V82I substitution at the second analysis, were seen in individual clones. Neither was detected at a subsequent time point. While these amino acid substitutions may represent polymorphisms, they have also been observed early in the course of nelfinavir treatment failure. Again, the detection of early drug resistance mutations did not represent impending treatment failure. Therefore, while PI-resistant variants may arise at plasma virus levels below 50 copies/ml in the presence of drug-selective pressure, drug-resistant variants do not become predominant or accumulate additional mutations when plasma virus levels are maintained below 50 copies/ml.
On the basis of the results of this study, we propose that every day a small subset of the cells within the latent reservoir become activated through an encounter with an antigen or another activating stimuli and that the virus released by these cells contributes to the low-level viremia. These viruses may undergo some additional rounds of replication. However, our results suggest that this additional replication is limited sufficiently that resistance mutations do not accumulate. This model explains why the reservoir for HIV-1 in resting CD4+
T cells does not decay with prolonged HAART even though there is continuous activation of cells in the reservoir. When additional rounds of replication are occurring, the number of latently infected cells that must be activated to fuel this replication may be such a small fraction of the reservoir that decay will not be significant even over a time scale of years (40
). Using an exponential decay model (31
), we calculate that the number of productively infected cells required to give rise to plasma virus levels of between 5 and 50 copies per ml in a child weighing 30 kg (estimated total extracellular fluid volume, 6 liters) ranges between 1,000 and 10,000 cells, respectively. This calculation assumes that N
, the number of virions produced per productively infected cell, is 1,000 virions (21
), and that the clearance rate constants c
, for plasma virions, and δ, for productively infected CD4+
T cells, are 23 day−1
and 0.7 day−1
, respectively (29
). If all of these productively infected cells were to be generated by the activation of cells in the latent reservoir and if all of the activated cells were to die, then decay of the reservoir would be expected. Because the reservoir is stable, it is necessary to postulate that not all of the productively infected cells come from the reservoir. Our model suggests that virus released by cells reactivated from latency may infect some other cells secondarily. The stability of the reservoir could also reflect the process of proliferative renewal that maintains the memory T-cell compartment at roughly constant levels throughout life (42
). In this case, proviral DNA is preserved by cell division and should show genetic stability consistent with the previous observations reported by Ruff et al. on the stability of this reservoir in children (38
). It is also possible that the stability of the reservoir reflects survival of some latently infected cells after a cytokine-induced state of partial activation in which the cells become permissive for some level of virus production but do not die as quickly as antigen-stimulated cells. Indeed, in vitro studies have shown resting CD4+
T cells can be partially activated by cytokine signals to result in HIV-1 production (44
). Infected cells may be more likely to persist during antiretroviral treatment due to waning cytotoxic T lymphocyte responses (2
). Finally, if the burst size is significantly greater (for example, 50,000), then the activation of fewer than 200 latently infected cells per day would give rise to a plasma virus level of 50 copies/ml. At this rate of activation, only limited decay of the latent reservoir would be expected. Thus, there are several possible mechanisms that would allow the pool of latently infected cells to contribute to ongoing viremia without showing substantial decay.
In summary, the data presented above provide evidence that during effective HAART, HIV-1 infection is dynamic, with the persistent production of virus that is sensitive to the PIs and that is related to latent HIV-1 in resting CD4+
T cells. This dynamic model of HIV-1 infection during suppressive HAART is consistent with recent studies of the suppressed state in HIV-1-infected adults (8
). Understanding the contribution of viral reservoirs to the fueling of virus replication during effective HAART in children is important for targeting future therapeutic strategies for HIV-1 infection. Furthermore, knowledge of the clinical implications of low levels of plasma virus during HAART is of paramount importance as more sensitive plasma HIV-1 RNA assays are incorporated into care and are used to guide therapeutic decisions.