Eradication of the HIV-1 latent reservoir represents the current paradigm to developing a cure for AIDS. HIV-1 has evolved multiple mechanisms to evade CD8 T cell responses, including HIV-1 Nef–mediated downregulation of MHC-I from the surface of infected cells. Nef transcripts and protein are detectable in samples from aviremic donors, suggesting that Nef expression in latently HIV-1–infected CD4 T cells protects them from immune-mediated clearance. Here, we tested 4 small molecule inhibitors of HIV-1 Nef in an in vitro primary CD4 T cell latency model and measured the ability of autologous ex vivo or HIV-1 peptide–expanded CD8 T cells to recognize and kill latently infected cells as a function of inhibitor treatment. Nef inhibition enhanced cytokine secretion by autologous CD8 T cells against latently HIV-1–infected targets in an IFN-γ release assay. Additionally, CD8 T cell–mediated elimination of latently HIV-1–infected cells was significantly enhanced following Nef blockade, measured as a reduction in the frequency of infected cells and Gag protein in cultures following viral outgrowth assays. We demonstrate for the first time to our knowledge that Nef blockade, in combination with HIV-specific CD8 T cell expansion, might be a feasible strategy to target the HIV-1 latent reservoir that should be tested further in vivo.
Shariq Mujib, Aamir Saiyed, Saleh Fadel, Ardalan Bozorgzad, Nasra Aidarus, Feng Yun Yue, Erika Benko, Colin Kovacs, Lori A. Emert-Sedlak, Thomas E. Smithgall, Mario A. Ostrowski
A schematic illustrating the study assays as described in the Methods section is shown. Latently infected HIV-1 CD4 T cells were generated in vitro from PBMC donated by an HIV-1–infected donor in a CCL19 dependent model and cultured with inhibitors targeting HIV-1 Nef. In parallel, PBMC from the same donor were stimulated with HIV-1 Gag and Nef peptide pools to expand HIV-specific CD8 T cells over a 6-day period. CD8 T cells were then isolated from these peptide-stimulated cultures or unstimulated, freshly thawed PBMC and cocultured with CD4 T cells in a minimal culture medium overnight in an IFN-γ release assay. In some experiments, following 18 hours, virus production was induced by the addition of IL-15. An MHC-I blocking antibody was added to cultures to prevent further CD8 T cell–mediated killing of HIV-1–infected CD4 T cells. HIV-1 Gag p24 was quantified via ELISA in the supernatant, as well as by intracellular flow cytometry, as a measure of CD8 T cell elimination of HIV-1–infected cells HAART was maintained in the cultures throughout to repress additional viral replication.