CELL-ASSOCIATED AND CELL-FREE VIRUS DURING HAART

Residual HIV-1 Provirus In Vivo during HAART

Persistently infected, nonactivated CD4⁺ T cells have been demonstrated in the peripheral blood of HIV-1-infected individuals, after treatment effectively suppresses most productive viral infec- tion.²⁸ Replication-competent viruses can be recovered from these proviral-positive cells after CD8⁺ T cell depletion in vitro.^29-31 The vast majority of these viruses are CCR5-tropic.³² In addition, this cell reservoir is established soon after primary HIV-1 infection³³ and can be activated by proinflam- matory cytokines in vitro and potentially in vivo.³⁴ Suppressive HAART initiated before primary HIV-1 seroconversion and during perinatal HIV-1 infection has also been shown to be unable to halt the development of this replication-competent virus reservoir in CD4⁺ T cells.^35,36

This replication-competent but latent HIV-1 pro viral DNA resides in resting memory or CD45RO CD4⁺ T lymphocytes and also in resting naive or CD45RA CD4⁺ T lymphocytes, albeit at lower levels.³² It has been 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.³² Given that infected resting memory and naive CD4⁺ T lymphocytes harbor replication-competent proviral DNA, it is not surprising that viral rebound could occur for most, if not every, patient at some time after discontinuation of HAART, as these cells turn over slowly.

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.²⁹ As resistance mutations in the reverse transcriptase (RT) and protease (PR) genes of HIV-1 are correlated with ongoing viral replication despite treatment,³⁷ this finding suggests that these viral strains may represent “archival” species from a time before treatment.

Cell-Associated Virus

It was hypothesized that persistently infected CD4⁺ T cells containing nondefective 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 with 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, modest evolution of viral envelope sequences occurred over time.¹¹ Other studies demonstrated that during what seems 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 evolution of viral sequences in cellular reser- voirs.⁴⁴,⁴⁵ These “archival” viruses did not demonstrate antiretroviral resistance mutations, even in those strains that could undergo low-level replication.

In studies by Furtado et al.⁴⁶ and recently confirmed by another group,⁴⁷ 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 preintegration 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 was suggested,⁴⁷ further studies are necessary to confirm this finding.

In one study,⁴⁴ a further complexity toward characterizing HIV-1 persistence during HAART was also demonstrated, as certain patients had cells with multiply spliced viral 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 nonproductive infection. These patterns of viral RNA species expression may be based on the state of host cell activation. Thus, several forms of cryptic replication might “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 people 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.^1,52,53 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.⁵⁰

Most,^54,55 but possibly not all,⁵⁶ patients rebound rapidly with high levels of plasma viral RNA when standard suppressive HAART is discontinued, even when fewer than 50 copies/ml of plasma viral RNA are demonstrated for significant time periods.

Cell-Free Virus

Using a laboratory-based reverse transcriptase-polymerase chain reaction (RT-PCR) assay that can quantify at least 5 copies/ml of plasma viral RNA,⁵⁹ 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).⁶⁰ Of importance, 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 virions³⁹ in residual plasma HIV-1 load are still unknown and remain important for understanding possible viral spread within an infected host.

The viral decay characteristics in cells are different from decay characteristics in plasma.² In PBMCs, the first phase represents decay of productively infected and long-lived cells, whereas the second phase is decay of cell-associated viral DNA and mRNA. Viral reservoir decay characteristics in one analysis suggested that 60 years will be necessary for patients treated with suppressive HAART to continue therapy for potential viral eradication.⁶¹ Unfortunately, even this estimate may not represent a best-case scenario for patients, as this analysis did not fully take into account the low-level viral replication that occurs in many patients on HAART.

Some patients with fewer 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,⁶² which carry risks of viral resistance evolution.³⁸ The mean decay half-life of latent replication-competent proviral reservoirs is longer in patients with transient plasma viral RNA spikes than in those patients who consistently maintain plasma HIV-1 RNA levels with fewer than 50 copies/ml.⁶² A recent small study suggested that low-level spikes of plasma HIV-1 RNA in patients on previously suppressive HAART do not seem to associate with higher rates of viral rebound, at least in the short term,⁶³ but other preliminary studies showed lower CD4⁺ T cell counts in patients with plasma HIV-1 RNA spikes.^64,65 It will be important to determine whether these viral spikes are due to insufficient plasma drug levels and depressed drug penetration into unique reservoir sites or just continued HIV-1 replication in the lymphoid tissue and peripheral blood cells.

The findings that 60 to 75 years of suppressive HAART would be necessary to possibly allow latent reservoir eradication were suggested to be due more to the slow decay of a truly latent proviral state rather than to ongoing viral replication.⁶¹ 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 and more than 50 copies/ml of plasma viral RNA on HAART may allow viral eradication in this select group of patients in a shorter time period.⁶² Nonetheless, this possibility assumes that even in these patients, there exist no other viral reservoirs in peripheral blood, lymphoid tissue, or other solid organs¹⁴ and that subclinical viral replication did not occur.