HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir

Abstract

The persistence of latently infected, resting CD4+ T cells is considered to be a major obstacle in preventing the eradication of HIV-1 even in patients who have received effective antiviral therapy for an average duration of 5 years. Although previous studies have suggested that the latent HIV reservoir in the resting CD4+ T cell compartment is virologically quiescent in the absence of activating stimuli, evidence has been mounting to suggest that low levels of ongoing viral replication persist and in turn, prolong the overall half-life of HIV in patients receiving antiviral therapy. Here, we demonstrate the persistence of replication-competent virus in CD4+ T cells in a cohort of patients who had received uninterrupted antiviral therapy for up to 9.1 years that rendered them consistently aviremic throughout that time. Surprisingly, substantially higher levels of HIV proviral DNA were found in activated CD4+ T cells when compared with resting CD4+ T cells in the majority of patients we studied. Phylogenetic analyses revealed evidence for cross infection between the resting and activated CD4+ T cell compartments, suggesting that ongoing reactivation of latently infected, resting CD4+ T cells and spread of virus by activated CD4+ T cells may occur in these patients. Such events may allow continual replenishment of the CD4+ T cell reservoir and resetting of the half-life of the latently infected, resting CD4+ T cells despite prolonged periods of aviremia.

Figure 1

Plasma viremia and CD4⁺ and CD8⁺ T cell counts of 4 representative study participants. The dotted lines indicate the limit of detection of plasma viremia (<500 or <50 copies of HIV RNA per ml).

Figure 2

Frequency of replication-competent HIV in CD4⁺ T cells of infected individuals receiving effective antiviral therapy for prolonged periods of time. The frequency of cells carrying infectious HIV (y axis) was assessed by activation of purified CD4⁺ T cells from the patients studied (x axis) with replicates of 1 × 10⁶ CD4⁺ T cells per well in 12-well plates.

Figure 3

Persistence of HIV in resting and activated CD4⁺ T cells of infected individuals receiving effective antiviral therapy for prolonged periods of time. (A) Frequency of FACS-sorted resting and activated CD4⁺ T cells carrying HIV proviral DNA. The median values are shown as black bars. (B) Levels of cell-free virions released by resting and activated CD4⁺ T cells in overnight cultures in the absence of activating stimuli. Cell-free supernatants from each culture harvested after 1 day of culture were subjected to the Amplicor HIV-1 test (detection limit of 50 copies of HIV RNA per ml).

Figure 4

Phylogenetic analysis of HIV env DNA and evidence for cross infection between resting and activated CD4⁺ T cells in patients receiving effective antiviral therapy. (A) Phylogenetic trees of HIV env sequences in resting (blue circles) and activated (red circles) CD4⁺ T cells of 2 representative patients. The outgroup sequences were obtained from unrelated HIV-infected patients. The direction of HIV migration from activated to resting (pink arrow) and from resting to activated (blue arrow) CD4⁺ T cells is shown. The bar indicates genetic distance. (B) The number of viral migration events observed on the phylogenetic trees within the resting and activated CD4⁺ T cell compartments is shown.