An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression

Abstract

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.

Bovine leukemia virus: current perspectives

“…[78][79][80] It would seem that the combination of drugs provides a more effective reactivation and elimination of the persistently infected cells. 81,82 Nevertheless, a more achievable approach would be to better identify the main mechanism(s) causing viral latency, and improve our knowledge on the relative contribution of each of the silencing mechanisms involved. In this way, it will be feasible to target and eliminate the latent virus reservoir with the appropriated drug(s).…”

Molecular mechanisms of HIV latency

“…Combinations of PKC or MAPK agonists and LRAs will be needed to mitigate toxic effects and lower effective doses (73,74,91). Such LRAs include HDACis, BETis, and 5′ azacytidine, among others.…”

“…Such LRAs include HDACis, BETis, and 5′ azacytidine, among others. Indeed, vorinostat, romidepsin, panobinostat, and JQ1 act synergistically with PKC agonists to reactivate HIV transcription (43,(91)(92)(93)(94)(95). Whereas PKC agonists activate NF-κB and increase overall levels of P-TEFb and CycL/CDK11, LRAs primarily affect the P-TEFb equilibrium.…”

“…Thus, LRAs limit the concomitant production of cytokines induced by PKC agonists. Most importantly, lower concentrations of PKC agonists and LRAs can be used in combination to achieve optimal HIV reactivation (73,91). Time of administration might also be an important therapeutic component, as PKC agonists and LRAs act through different mechanisms.…”

Recent developments in the effort to cure HIV infection: going beyond N = 1

“…One of the major problems in the search for effective LRAs is that, despite impressive activity in various in vitro models, most LRAs, including the HDAC inhibitors, have weak activity in ex vivo studies using resting CD4 + T cells from patients on ART (74). Fortunately, some combinations of LRAs are now beginning to show levels of latency reversal comparable to global T cell activation (75,76). In clinical trials, no reduction in the reservoir has yet been demonstrated, but there is evidence for increases in cell-associated HIV RNA and for slight transient increases in plasma HIV RNA with certain HDAC inhibitors (17,18).…”