Synergistic latency reversal by the SMAC mimetic AZD5582 and iBET JQ1, combined with Nef ablation, facilitates immune-mediated elimination of latently HIV-1-infected T-cells

Recent breakdowns in the supply chains of antiretroviral therapy (ART) to lower income countries, where they are needed most, underpin the pressing need for an accessible and scalable HIV cure that would allow ART-free control of the virus. The ‘shock-and-kill’ cure concept shows promise in its ability to reactivate HIV-1 transcription in latently infected cells in vivo in people living with HIV-1 but has failed to result in a meaningful reduction of the size of the reactivatable viral reservoir. We therefore hypothesised that the efficiency of reversal of transcriptional quiescence, and the resulting HIV-1 antigen expression and presentation, may be insufficient to achieve full immunological visibility of HIV-1-infected cells. To gain a deeper understanding of potential LRA-specific shortcomings, we developed a novel model of HIV-1 latency - Jurkat E6.1 subclonal cell lines, each harbouring a single, full-length, NL4.3-based provirus with a GFP _OPT reporter inserted into the V5 loop of the viral Env protein. Using this model, we quantified HIV-1 reactivation, as well as expression and surface presentation of Env after treatment with a panel of known latency reversal agents (LRAs) and their synergistically-acting combinations. HIV-1 reactivation with the PKC agonist Bryostatin-1 limited the cell-surface presentation of viral Env, a phenomenon we found being linked to Bryostatin-1’s ability to induce the expression of the restriction factor GBP5 in T-cell lines and primary CD4+ T-cells, and induced a cellular state which was resistant to apoptosis. A combined treatment of Bryostatin-1 and the BET inhibitor (iBET) JQ1 resulted in synergistic HIV-1 reactivation and boosted levels of cell-surface Env, but failed to reduce resistance to apoptosis. In contrast, the combination of the SMAC mimetic (SMACm) AZD5582 and JQ1 markedly boosted cell-surface Env levels, without co-induction of GBP5, T-cell activation or apoptotic resistance. Crucially, HIV-1 Nef was found to be a potent antagonist of the cytotoxic killing of reactivating cells, most likely by its ability to inhibit apoptosis. Nef knockout, however, when paired with the AZD/JQ1 combination, displayed highest potency in inducing immune-mediated elimination of latently infected T-cells and presents a promising new approach for HIV cure programs.