Reliable Estimation of CD8 T Cell Inhibition of In Vitro HIV-1 Replication

Front Immunol. 2021 Jun 30:12:666991. doi: 10.3389/fimmu.2021.666991. eCollection 2021.


The HIV-1 viral inhibition assay (VIA) measures CD8 T cell-mediated inhibition of HIV replication in CD4 T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. The VIA has multiple sources of variability arising from in vitro HIV infection and co-culture of two T cell populations. Here, we describe multiple modifications to a 7-day VIA protocol, the most impactful being the introduction of independent replicate cultures for both HIV infected-CD4 (HIV-CD4) and HIV-CD4:CD8 T cell cultures. Virus inhibition was quantified using a ratio of weighted averages of p24+ cells in replicate cultures and the corresponding 95% confidence interval. An Excel template is provided to facilitate calculations. Virus inhibition was higher in people living with HIV suppressed on antiretroviral therapy (n=14, mean: 40.0%, median: 43.8%, range: 8.2 to 73.3%; p < 0.0001, two-tailed, exact Mann-Whitney test) compared to HIV-seronegative donors (n = 21, mean: -13.7%, median: -14.4%, range: -49.9 to 20.9%) and was stable over time (n = 6, mean %COV 9.4%, range 0.9 to 17.3%). Cross-sectional data were used to define 8% inhibition as the threshold to confidently detect specific CD8 T cell activity and determine the minimum number of culture replicates and p24+ cells needed to have 90% statistical power to detect this threshold. Last, we note that, in HIV seronegative donors, the addition of CD8 T cells to HIV infected CD4 T cells consistently increased HIV replication, though the level of increase varied markedly between donors. This co-culture effect may contribute to the weak correlations observed between CD8 T cell VIA and other measures of HIV-specific CD8 T cell function.

Keywords: CD4; CD8; HIV; JRCSF; ROC; T-cell; VIA; p24.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antiviral Agents / therapeutic use
  • CD4-Positive T-Lymphocytes / immunology
  • CD8-Positive T-Lymphocytes / immunology*
  • Case-Control Studies
  • Cells, Cultured
  • Coculture Techniques
  • Cross-Sectional Studies
  • HIV Core Protein p24 / immunology
  • HIV Seropositivity / blood
  • HIV Seropositivity / drug therapy
  • HIV Seropositivity / immunology*
  • HIV Seropositivity / virology
  • HIV-1 / immunology*
  • Host Microbial Interactions / immunology*
  • Humans
  • Treatment Outcome
  • Virus Replication / immunology*


  • Antiviral Agents
  • HIV Core Protein p24