Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy

Abstract

Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.

Figure 1

Quantitative analysis of integrated HIV-1 DNA in resting CD4⁺ T cells. (A) Detection of integrated HIV-1 DNA by using Alu-LTR PCR. Genomic DNA from resting CD4⁺ T cells was serially diluted 5-fold starting with 200,000 cell DNA equivalents and subjected to PCR in duplicate by using nested 5′ primers from conserved Alu and 3′ primer from conserved HIV-1 LTR sequences. This dilution amplifies both cellular DNA upstream of the integration site and integrated HIV-1 LTR. An aliquot of the diluted first PCR product was further subjected to the second round of PCR by using nested HIV-1 LTR-specific primers. (B) Genomic DNA from ACH-2 and U1 cells carrying one and two copies of an integrated form of HIV-1, respectively, was serially diluted and subjected to Alu-LTR PCR followed by a second round of the nested PCR. As negative controls, first PCR-DNA was used for the second round of PCR and a plasmid mimicking unintegrated DNA was subjected to two rounds of PCR in the same manner. (C) Representative results of Alu-LTR PCR of genomic DNA from purified resting CD4⁺ T cells from treated patients. The limiting dilution Alu-LTR PCR was carried out on the indicated number of cell equivalents of donor DNA. The product of two rounds of PCR was resolved in an agarose gel and bands were detected by Southern blot analysis by using an LTR-specific probe.

Figure 2

Frequencies of resting CD4⁺ T cells carrying various forms of HIV-1 DNA in patients receiving HAART with undetectable and detectable plasma virus and drug naive patients. (A) Frequencies of resting CD4⁺ T cells carrying total and integrated HIV-1 DNA. The copy numbers of HIV-1 DNA including both unintegrated and integrated HIV-1 DNA in purified resting CD4⁺ T cells in patients were calculated as described in the text. The frequencies of resting CD4⁺ T cells with integrated HIV-1 DNA was determined by Alu-LTR PCR by using serially diluted genomic DNA from resting CD4⁺ T cells (Fig. 1). (B) Frequencies of resting CD4⁺ T cells carrying replication-competent HIV-1 DNA were determined by activating purified resting CD4⁺ T cells on day 0. Frequencies of resting CD4⁺ T cells carrying persistent replication-competent HIV-1 DNA were determined by activating 6 day pre-incubated cells in the absence of activating stimuli. For each assay, a statistical method was used by Myers et al. (25) to calculate copy numbers or infectious units per million resting CD4⁺ T cells. The geometric mean frequencies and corresponding 95% confidence intervals for each data set are plotted to the right of the individual donor values. The letters a, b, and c in each panel refer to the three separate groups of patients: a, HAART treated, <500 copies HIV RNA/ml; b, HAART treated, >500 copies HIV RNA/ml; c, untreated.