Measuring the Frequency of Latent HIV-1 in Resting CD4⁺ T Cells Using a Limiting Dilution Coculture Assay

Methods Mol Biol. 2016;1354:239-53. doi: 10.1007/978-1-4939-3046-3_16.


Combination antiretroviral therapy (cART) can reduce HIV-1 viremia to clinically undetectable levels. However, replication competent virus persists in a long-lived latent reservoir in 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.

Keywords: HIV-1; Latency; Latent reservoir; Viral outgrowth assay.

MeSH terms

  • Anti-HIV Agents / therapeutic use*
  • CD4-Positive T-Lymphocytes / virology*
  • Cells, Cultured
  • Coculture Techniques / methods*
  • HIV Infections / drug therapy
  • HIV-1 / drug effects
  • HIV-1 / physiology*
  • Humans
  • Virus Cultivation / methods
  • Virus Latency* / drug effects


  • Anti-HIV Agents