Measuring inhibition of HIV replication by ex vivo CD8+ T cells

• We optimized an assay to determine suppression of viral replication in vitro.
• Our assay uses ex vivo effector T cells rather than cell lines.
• Background suppression levels are very low.
• Low levels of inhibition such as those induced by vaccination can be measured.

HIV replication is unrestrained in vivo in the vast majority of infected subjects, and the ability of some rare individuals to control this virus is poorly understood. Standard immunogenicity assays for detecting HIV-1-specific CD8+ T-cell responses, such as IFN-γ ELISpot and intracellular cytokine staining, generally fail to correlate with in vivo inhibition of HIV replication. Several viral inhibition assays, which measure the effectiveness of CD8+ T-cell responses in suppressing HIV replication in vitro, have been described; but most depend on in vitro expansion of CD8+ T cells, and some show inhibitory activity in HIV-negative individuals. We have optimized an assay to assess the suppressive capability of CD8+ T cells directly ex vivo, eliminating the potential for altering their function through activation or expansion prior to assay setup, and thereby enhancing the assay's sensitivity by avoiding non-specific inhibition. With this method, the ability of ex vivo CD8+ T cells to control HIV-1 replication in vitro can be quantified over several orders of magnitude. Specifically, our assay can be used to better define the antiviral function of CD8+ T cells induced by vaccination, and can provide insight into their ability to control viral replication if HIV infection occurs post-vaccination.