Selection of effective LRAs using a newly designed in vitro HIV latency reactivation protocol: toward future application in HIV samples

Abstract

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) persists in latent reservoirs, mainly within CD4+ T cells, which are refractory to antiretroviral therapy, and can lead to rapid viral rebound upon treatment interruption. The “shock and kill” strategy aims to eliminate latent reservoirs by inducing viral transcription through latency-reversing agents (LRAs), thereby exposing infected cells to immune-mediated clearance. We developed and validated a simple, low-cost, and highly reproducible in vitro screening protocol to evaluate the efficacy and safety of LRAs, using a two-color flow cytometry assay on ACH2 cells, a well-characterized model of HIV-1 latency. Using this method, we identified PEP005 and CUDC-907 as the most potent LRAs across multiple experimental settings. Their reactivation capacity was further confirmed through transcriptomic analysis, which revealed a significant upregulation of viral RNA copies following stimulation. In addition, we collaborated with Dompé for using the Exscalate platform, an innovative computer-aided drug discovery approach, together with the experimental validation, which led to the identification of Tandutinib, a tyrosine kinase and mTOR inhibitor, as a novel LRA candidate with appreciable latency-reversing activity in the ACH2 model. While the ACH2 cell line does not fully recapitulate the complexity of HIV latency in vivo, it offers a robust and scalable system for early-stage screening and prioritization of candidate LRAs. Importantly, the future application of these LRAs in ex vivo samples derived from people living with HIV, with a particular focus on pediatric samples, will be crucial to deepen our understanding of latency reactivation in clinically relevant settings and age-specific immune environments. IMPORTANCE This study addresses a major obstacle to curing human immunodeficiency virus type 1 (HIV-1) infection: the persistence of latent viral reservoirs that are not eliminated by current therapies. We developed a simple and reproducible assay to identify compounds capable of reactivating latent virus, a key step in cure strategies. Using this approach, we identified effective latency-reversing compounds and, through collaboration with Dompé using the EXSCALATE platform, we also identified Tandutinib as a promising new candidate for further investigation. This study addresses a major obstacle to curing human immunodeficiency virus type 1 (HIV-1) infection: the persistence of latent viral reservoirs that are not eliminated by current therapies. We developed a simple and reproducible assay to identify compounds capable of reactivating latent virus, a key step in cure strategies. Using this approach, we identified effective latency-reversing compounds and, through collaboration with Dompé using the EXSCALATE platform, we also identified Tandutinib as a promising new candidate for further investigation.