Abstract
HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4+ T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4+ T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4+ T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research.
Highlights
Treatment of HIV-1 infection with highly active antiretroviral therapy (HAART) can reduce plasma HIV-1 RNA levels in treated patients to below the detection limit of clinical assays (50 copies of HIV-1 RNA/ml) [1,2,3]
To determine whether HIV-1 DNA levels in peripheral blood mononuclear cells (PBMC) could be used as a surrogate measure of the size of the latent reservoir, we examined the correlation between results of the viral outgrowth assay on purified resting CD4+ T cells and the droplet digital polymerase chain reaction (PCR) assay for HIV-1 DNA in PBMC from the same blood sample
We examined whether the correlation between the results of the viral outgrowth and PCR assays might be improved if HIV1 DNA levels were measured in purified resting CD4+ T cells instead of unfractionated PBMC
Summary
Treatment of HIV-1 infection with highly active antiretroviral therapy (HAART) can reduce plasma HIV-1 RNA levels in treated patients to below the detection limit of clinical assays (50 copies of HIV-1 RNA/ml) [1,2,3]. The effective suppression of viremia initially encouraged hopes that the virus could be eradicated with two to three years of HAART [3]. A small fraction of resting memory CD4+ T cells carry integrated viral genomes. These cells do not produce virus particles while in the resting state, but can give rise to replication-competent virus following cellular activation [4,5]. These latently infected cells are rare but stable, even in patients on prolonged HAART [6,7,8,9,10,11]. Interruption of HAART leads to a rebound in viremia [12,13], typically from an Author Summary
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