Date Published: May 30, 2013
Publisher: Public Library of Science
Author(s): Gregory M. Laird, Evelyn E. Eisele, S. Alireza Rabi, Jun Lai, Stanley Chioma, Joel N. Blankson, Janet D. Siliciano, Robert F. Siliciano, Guido Silvestri.
HIV-1 persists in infected individuals in a stable pool of resting CD4+ T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4+ T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4+ T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.
Highly active antiretroviral therapy (HAART) has significantly reduced the morbidity and mortality associated with HIV-1 infection. However, while HAART can reduce plasma viral load to below the clinical limit of detection (50 copies HIV-1 RNA/mL) in adherent patients –, this treatment is not curative. Even in individuals on prolonged suppressive HAART, HIV-1 persists as a latent but replication-competent provirus integrated in the genomes of a small percentage of resting memory CD4+ T cells –. These latently infected cells are extremely long lived as a consequence of the biology of memory T cells, with an estimated half-life of 44 months , . The extreme stability of this HIV-1 reservoir precludes eradication with HAART alone and suggests that, without disruption of this reservoir, infected individuals must remain on HAART for the remainder of their lives , .
Latent HIV-1 infection of resting CD4+ T cells remains the major barrier to HIV-1 eradication. A number of small molecules have been identified that are capable of reactivating transcription of otherwise silent HIV-1 proviruses –, . Some of these compounds have already entered clinical trials , and drug discovery efforts to find additional compounds that can perturb or eliminate latent HIV-1 continue. Concurrently, immunological approaches are being investigated and have shown promise . However, a key hurdle facing HIV-1 eradication efforts has, until recently, been largely ignored: the development of a reliable and simple assay to measure the size of the HIV-1 latent reservoir. Such an assay is absolutely required for evaluating the effectiveness of an eradication strategy. PCR based assays are being used to quantify proviruses in T cell subsets and the level of residual viremia in HIV-1 infected patients , –. A recent study has compared results of various PCR based assays with those obtained with the viral outgrowth assay using a set of samples from two well characterized cohorts of patients on HAART . Because current PCR assays detect both replication-competent and defective proviruses, the correlation between infected cell frequencies measured by PCR and viral outgrowth was not strong, with the exception of an assay measuring integrated HIV-1 DNA in PBMC . The measurement of integrated HIV-1 DNA by Alu PCR  is of particular interest because the stable reservoir for HIV-1 consists of resting CD4+ T cells harboring integrated HIV-1 DNA , . It is likely that this and other PCR based assays will play an important complementary role to viral outgrowth assays. Prior to the present study, the standard viral outgrowth assay was the only assay available to directly quantify the frequency of resting CD4+ T cells harboring latent but replication-competent viral genomes.