persists in a latent reservoir (LR) despite antiretroviral therapy (ART)1-5. none of the latency reversing agents (LRAs) tested induced outgrowth of HIV-1 from the LR of patients on ART. Using a novel RT-qPCR assay specific for all HIV-1 mRNAs we demonstrate that LRAs that do not cause T cell activation do not induce significant increases in intracellular HIV-1 mRNA in patient cells; only the PKC agonist bryostatin-1 caused substantial increases. These findings demonstrate that current models do not fully recapitulate mechanisms governing HIV-1 latency when administered individually. HIV-1 cure is hindered by viral persistence in a small fraction (~1/106) of resting CD4+ T cells (rCD4s) that harbor latent but replication-competent proviruses1-3. Upon cellular activation latency is reversed and replication-competent virus is produced. Although T cell activation reverses latency global T cell activation is toxic generating interest in small molecule latency-reversing agents (LRAs) that do not activate T cells. Due to the low frequency of latently infected rCD4s cell models have been used to identify a number Rabbit Polyclonal to Caspase 7 (p11, Cleaved-Ala207). of mechanistically distinct LRAs. These include: (1) histone deacetylase Baicalin (HDAC) inhibitors thought to function through epigenetic and other mechanisms11-14; (2) disulfiram postulated to involve nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)15 16 and (3) the bromodomain-containing protein 4 (BRD4) inhibitor JQ1 which elicits effects through positive transcription elongation factor (P-TEFb)17-20. Acting through signaling pathways associated with T cell activation protein kinase C (PKC) agonists such as phorbol esters prostratin21-23 and bryostatin-112 24 also reverse latency Baicalin in cell models. Evidence that putative LRAs reverse latency in primary rCD4s from HIV-1-infected individuals is limited; disulfiram and the HDAC inhibitor vorinostat have been tested in patient cells with inconsistent Baicalin results11 13 16 27 28 Clinical trials in patients on ART are ongoing with disulfiram and the HDAC inhibitors vorinostat romidepsin and panobinostat27 29 A recent trial of disulfiram showed no consistent evidence of latency reversal30. In another clinical trial a single dose of vorinostat modestly increased intracellular RNAs containing HIV-1 sequences in rCD4s of patients on ART27. treatment of patient cells with vorinostat induced outgrowth in some studies11 13 but no virion production in another study28. Importantly no LRA has been shown to reduce the size of the LR. A consistent validation strategy has not been employed to compare putative LRAs. Given the costs and risks associated with clinical trials such a strategy is important for HIV-1 eradication research. Therefore we utilized three independent assays to evaluate the efficacy of LRAs in cells from HIV-1 infected individuals on suppressive ART (participant characteristics in Supplementary Table 1). We first tested LRAs in a modified viral outgrowth assay1. In the original assay patient-derived rCD4s were activated and co-cultured with CD4+ T lymphoblasts from healthy donors to expand released virus. Induction of outgrowth provides conclusive evidence of latency reversal. In the modified assay T cell activation was replaced with LRA treatment. The subsequent co-culture of patient rCD4s with healthy donor lymphoblasts constitutes a mixed lymphocyte reaction which induces background reactivation of latent HIV-131 and complicates LRA evaluation. Therefore we treated rCD4s with LRAs and then cultured the cells with a transformed CD4+ T cell line (MOLT-4/CCR5) (Fig. 1a) that supports robust HIV-1 replication but does not induce allogeneic stimulation of rCD4s (Supplementary Fig. 1a-c). We treated five million purified rCD4s from infected individuals on ART with single LRAs for 18 h and then co-cultured the cells with MOLT-4/CCR5 cells for 14 days to permit viral outgrowth. T cell activation with phorbol 12-myristate 13-acetate + ionomycin (PMA/I) served as a positive control. We concurrently measured the frequency of latently infected cells32. We evaluated vorinostat romidepsin panobinostat disulfiram and bryostatin-1 at clinically relevant concentrations that effectively reversed latency in a primary cell model (see below) and that were not toxic to rCD4s. No drug treatment induced cell death as shown by the lack of 7-AAD staining (Fig. 1b). Surprisingly none of the LRAs induced viral outgrowth from cells from any individual tested while PMA/I-treated.