Measuring the frequency of latent HIV-1 in resting CD4⁺ T cells using a limiting dilution coculture assay

Combination antiretroviral therapy (cART) can reduce HIV-1 viremia to clinically undetectable levels. However, replication competent virus persists in a long-lived latent reservoir n resting, memory CD4⁺ T cells. The latent reservoir in resting CD4⁺ T cells is the major barrier to curing HIV-1 infection. The recent case of the Berlin patient has suggested that it may be possible to cure HIV-1 infection in certain situations. As efforts to cure HIV-1 infection progress, it will become critical to measure the latent reservoir in patients participating in clinical trials of eradication strategies. Our laboratory has developed a limiting dilution virus outgrowth assay that can be used to demonstrate the presence and persistence of latent HIV-1 in patients. Here we describe both the original and a simplified version of the quantitative virus outgrowth assay (QVOA) to measure the frequency of latently infected resting CD4⁺ T cells with replication competent provirus in patients on suppressive cART.