Viral sanctuaries during highly active antiretroviral therapy in a nonhuman primate model for AIDS

Abstract

Highly active antiretroviral therapy (HAART) enables long-term suppression of plasma HIV-1 loads in infected persons, but low-level virus persists and rebounds following cessation of therapy. During HAART, this virus resides in latently infected cells, such as resting CD4(+) T cells, and in other cell types that may support residual virus replication. Therapeutic eradication will require elimination of virus from all reservoirs. We report here a comprehensive analysis of these reservoirs in fluids, cells, and tissues in a rhesus macaque model that mimics HAART in HIV-infected humans. This nonhuman primate model uses RT-SHIV, a chimera of simian immunodeficiency virus containing the HIV-1 reverse transcriptase (RT). Methods were developed for extraction, preamplification, and real-time PCR analyses of viral DNA (vDNA) and viral RNA (vRNA) in tissues from RT-SHIV-infected macaques. These methods were used to identify viral reservoirs in RT-SHIV-infected macaques treated with a potent HAART regimen consisting of efavirenz, emtricitabine, and tenofovir. Plasma virus loads at necropsy ranged from 11 to 28 copies of vRNA per ml. Viral RNA and DNA were detected during HAART, in tissues from numerous anatomical locations. Additional analysis provided evidence for full-length viral RNA in tissues of animals with virus suppressed by HAART. The highest levels of vDNA and vRNA in HAART-treated macaques were in lymphoid tissues, particularly the spleen, lymph nodes, and gastrointestinal tract tissues. This study is the first comprehensive analysis of the tissue and organ distribution of a primate AIDS virus during HAART. These data demonstrate widespread persistence of residual virus in tissues during HAART.

FIG. 1.

Experimental design for treatment and analysis of RT-SHIV-infected macaques.

FIG. 2.

Plasma virus RNA in RT-SHIV-infected rhesus macaques treated with efavirenz plus emtricitabine plus tenofovir. Plasma VLs in the nine individual animals of the drug-treated group are shown. The arrow at 6 weeks indicates the time that drug therapy was initiated, and the arrow at 32 weeks indicates the time at which drug therapy was stopped. The mean VL for the control group was obtained from a total of 13 RT-SHIV-infected macaques that received no HAART. A cross indicates the necropsy of a control animal and a reduction of the total number of animals in the group from that point forward. The dotted line indicates the lower limit of detection of the VL assay (50 copies of viral RNA per ml of plasma).

FIG. 3.

Preamplification of SIV and cellular IL-2 DNAs. SIV gag and genomic IL-2 (gIL-2) DNAs were preamplified by PCR for 5, 10, 15, 20, and 25 cycles. Quantitative PCR analysis was then performed on these samples. Each point represents the average C_T value for three replicates. The greatest standard deviation of any point was 0.28. Error bars are too small to be visible.

FIG. 4.

Biological variation of vDNA and vRNA levels in multiple samples of the same tissues. (A) vDNA; (B) vRNA. Four samples were taken from each of the following tissues of a single RT-SHIV-infected macaque (Mmu 36606; plasma VL, 4,300 copies vRNA per ml): spleen, a mesenteric lymph node (MLN), and an axillary lymph node (AxLN). Nucleic acid extraction, preamplification, and real-time PCR for measuring levels of vRNA and vDNA were performed independently for each tissue sample, in duplicate. Each bar represents the mean for two replicates, with an error bar for the standard deviation.

FIG. 5.

Analysis of viral transcripts in tissues. Axillary lymph nodes were obtained at necropsy from one macaque after rebound from HAART (Mmu 35913; plasma viral load, 930 copies/ml) and from another macaque during HAART (Mmu 35343; plasma viral load, 28 copies/ml). PCR amplification was performed to detect gag, pol, and env RNAs. A control reaction without RT (mock cDNA sample) was included for each animal. Lane M, Bionexus All Purpose Lo marker.

FIG. 6.

Enrichment of resting CD4⁺ T cells. MLN lymphocytes from a representative animal (Mmu 35389) were enriched for CD4⁺ T cells by negative magnetic bead selection. Additional HLA-DR beads were included to remove activated T cells. CD20 beads were added to remove B cells, as the original bead cocktail containing CD19 beads was ineffective. (A) Removal of CD8⁺ T cells after enrichment. (B) Removal of HLA-DR⁺ CD4⁺ T cells. Left panels show samples before enrichment; right panels show samples after enrichment.