A CD3/CD28 microbead-based HIV-1 viral outgrowth assay

Aims: Latently infected resting CD4 T cells represent a major barrier to HIV-1 eradication efforts. The standard assays used for measuring this reservoir induce activation of resting CD4 T cells with either phytohaemagglutinin (PHA) with irradiated feeder cells, or with anti-CD3 antibodies. We designed a study to compare the sensitivity of a new assay (based on the stimulation of CD4 T cells with anti-CD3 and anti-CD28 coated microbeads) with that of the traditional PHA- and feeder-based viral outgrowth assay. Methods: Resting CD4 T cells from 10 HIV-1-infected patients on suppressive combination antiretroviral therapy (cART) regimens were cultured in the traditional PHA/feeders viral outgrowth assay and the new CD3/CD28 bead-based assay. Flow cytometry was used to assess the kinetics of activation of resting CD4 T cells in the two different assays. Results: There was no significant difference in the sensitivity of the two assays. The median frequency of latently infected cells was 0.83 infectious units per million (IUPM) for the PHA/feeders assay and 0.54 IUPM with the CD3/CD28 bead-based assay. However, while virus was obtained from all 10 patients with the traditional PHA/feeders outgrowth assay, no virus was obtained from two of 10 patients with the novel anti-CD3/CD28 bead-based viral outgrowth assay (IUPM <0.02). Conclusion: The new CD3/CD28 bead-based assay has comparable sensitivity to the PHA/feeders assay and does not require the addition of feeders, making it a simpler and less labour-intensive alternative to the standard PHA/feeders-based assay.