The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs. Persistence of latently infected cells during cART is a major hurdle for HIV-1 eradication. A widely proposed strategy to purge these reservoirs involves the reactivation of latent proviruses. The low levels of active P-TEFb and the cytoplasmic sequestration of NF-κB in resting infected cells largely contribute to maintenance of HIV-1 latency. Therefore, utilization of chemical compounds that target both pathways may lead to more potent effects on HIV-1 reactivation than the effect mediated by the individual drug treatments. In this study, we showed that combined treatments of PKC agonists (prostratin, bryostatin-1 and ing-B) with compounds releasing P-TEFb (JQ1, I-BET, I-BET151 and HMBA) exhibited a synergistic increase in viral reactivation from latency. In-depth comparison of combined treatments in various in vitro cellular models of HIV-1 latency as well as in ex vivo primary cell cultures from cART-treated HIV⁺ aviremic patients identified bryostatin-1+JQ1 and ing-B+JQ1 to potently reactivate latent HIV-1. The potent effects of these two combinations were detected as early as 24 hours post-treatment. Importantly, bryostatin-1 was used at concentrations below the drug plasma levels achieved by doses used in children with refractory solid tumors. Our mechanistic data established a correlation between potentiated P-TEFb activation and potentiated or synergistic (depending on the HIV-1 latency cellular model used) induction of HIV-1 gene expression observed after the combined versus individual drug treatments. In conclusion, our results establish a proof-of-concept for PKC agonists combined with compounds releasing active P-TEFb as a strategy proposed for a cure or a durable remission of HIV infection.

One of the most explored therapeutic approaches aimed at eradicating HIV-1 reservoirs is the “shock and kill” strategy which is based on HIV-1 reactivation in latently-infected cells (“shock” phase) while maintaining antiretroviral therapy (ART) in order to prevent spreading of the infection by the neosynthesized virus. This kind of strategy allows for the “kill” phase, during which latently-infected cells die from viral cytopathic effects or from host cytolytic effector mechanisms following viral reactivation. Several latency reversing agents (LRAs) with distinct mechanistic classes have been characterized to reactivate HIV-1 viral gene expression. Some LRAs have been tested in terms of their potential to purge latent HIV-1 in vivo in clinical trials, showing that reversing HIV-1 latency is possible. However, LRAs alone have failed to reduce the size of the viral reservoirs. Together with the inability of the immune system to clear the LRA-activated reservoirs and the lack of specificity of these LRAs, the heterogeneity of the reservoirs largely contributes to the limited success of clinical trials using LRAs. Indeed, HIV-1 latency is established in numerous cell types that are characterized by distinct phenotypes and metabolic properties, and these are influenced by patient history. Hence, the silencing mechanisms of HIV-1 gene expression in these cellular and tissue reservoirs need to be better understood to rationally improve this cure strategy and hopefully reach clinical success.

With the development of effective combined anti-retroviral therapy (cART), there is significant reduction in deaths associated with human immunodeficiency virus type 1 (HIV-1) infection. However, the complete cure of HIV-1 infection is difficult to achieve without the elimination of latent reservoirs which exist in the infected individuals even under cART regimen. These latent reservoirs established during early infection have long life span, include resting CD4+ T cells, macrophages, central nervous system (CNS) resident macrophage/microglia, and gut-associated lymphoid tissue/macrophages, and can actively produce virus upon interruption of the cART. Several epigenetic and non-epigenetic mechanisms have been implicated in the regulation of viral latency. Epigenetic mechanisms such as histone post translational modifications (e.g., acetylation and methylation) and DNA methylation of the proviral DNA and microRNAs are involved in the establishment of HIV-1 latency. The better understanding of epigenetic mechanisms modulating HIV-1 latency could give clues for the complete eradication of these latent reservoirs. Several latency-reversing agents (LRA) have been found effective in reactivating HIV-1 reservoirs in vitro, ex vivo, and in vivo. Some of these agents target epigenetic modifications to elicit viral expression in order to kill latently infected cells through viral cytopathic effect or host immune response. These therapeutic approaches aimed at achieving a sterilizing cure (elimination of HIV-1 from the human body). In the present review, we will discuss our current understanding of HIV-1 epigenomics and how this information can be moved from the laboratory bench to the patient’s bedside.

Reactivation of HIV gene expression in latently infected cells together with an efficient cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing the reservoir size. Results from HIV clinical trials using deacetylase inhibitors (HDACIs) question the efficiency of these latency‐reversing agents (LRAs) used alone and underline the need to evaluate other LRAs in combination with HDACIs. Here, we evaluated the therapeutic potential of a demethylating agent (5‐AzadC) in combination with clinically tolerable HDACIs in reactivating HIV‐1 from latency first in vitro and next ex vivo . We showed that a sequential treatment with 5‐AzadC and HDACIs was more effective than the corresponding simultaneous treatment both in vitro and ex vivo . Interestingly, only two of the sequential LRA combinatory treatments tested induced HIV‐1 particle recovery in a higher manner than the drugs alone ex vivo and at concentrations lower than the human tolerable plasmatic concentrations. Taken together, our data reveal the benefit of using combinations of 5‐AzadC with an HDACI and, for the first time, the importance of treatment time schedule for LRA combinations in order to reactivate HIV. ![][1] Sequential administration of latency‐reversing agents (LRAs), namely deacetylase inhibitors (HDACIs) and demethylating agents, together with an efficient cART, could represent an adjuvant anti‐HIV‐1 therapy to induce viral production and possibly reduce the size of latent HIV‐1 reservoirs. EMBO Mol Med (2016) 8: 117–138 [1]: /embed/graphic-1.gif