A scalable RNA RT-PCR based assay to quantify latency disruption in HIV-1 infected cells from patients on antiretroviral therapy (VIR1P.1001)

Abstract

Abstract Antiretroviral therapy suppresses HIV-1 replication but virus persists in stable reservoirs. A facile way to quantify cells in such reservoirs is HIV DNA PCR, however most of these cells are unlikely to ever produce replication-competent virus. On the other hand, viral outgrowth assays provide virologic realism, but are resource intensive. Detection of RNA by RT-PCR provides some virologic realism while allowing for high throughput. We present a method in which flow cytometry purified CD4+ T cells from patients on antiretroviral therapy are placed in limiting dilution culture for 4 days. The cells are stimulated with antibodies to CD2, CD3, and CD28 and viral replication is inhibited with efavirenz. Viral RNA is purified from the culture media using a paramagnetic nanoparticle based method that is scalable for robotics. Real time quantitative RT-PCR is performed with an HIV gag primer and probe set. This assay detects as few as 47 copies of HIV RNA induced from a single cell from the ACH-2 cell line. Using resting memory CD4+ T cells from patients on antiretroviral therapy, the frequency of induced HIV RNA production varied from 1 in 40,000 cells to fewer than 1 in 1,000,000. There was no correlation between frequency of HIV RNA producing cells and frequency of proviral DNA. This assay represents a scalable method for quantifying the frequency of HIV producing cells from patients with viral suppression and for evaluating reservoir elimination strategies for such patients.