Latency reversal agents affect differently the latent reservoir present in distinct CD4+ T subpopulations

Abstract

Latency reversal agents (LRAs) have proven to induce HIV-1 transcription in vivo but are ineffective at decreasing the size of the latent reservoir in antiretroviral treated patients. The capacity of the LRAs to perturb the viral reservoir present in distinct subpopulations of cells is currently unknown. Here, using a new RNA FISH/flow ex vivo viral reactivation assay, we performed a comprehensive assessment of the viral reactivation capacity of different families of LRAs, and their combinations, in different CD4 + T cell subsets. We observed that a median of 16.28% of the whole HIV-reservoir induced HIV-1 transcripts after viral reactivation, but only 10.10% of these HIV-1 RNA + cells produced the viral protein p24. Moreover, none of the LRAs were powerful enough to reactivate HIV-1 transcription in all CD4 + T cell subpopulations. For instance, the combination of Romidepsin and Ingenol was identified as the best combination of drugs at increasing the proportion of HIV-1 RNA + cells, in most, but not all, CD4 + T cell subsets. Importantly, memory stem cells were identified as highly resistant to HIV-1 reactivation, and only the combination of Panobinostat and Bryostatin-1 significantly increased the number of cells transcribing HIV within this subset. Overall, our results validate the use of the RNA FISH/flow technique to assess the potency of LRAs among different CD4 + T cell subsets, manifest the intrinsic differences between cells that encompass the latent HIV reservoir, and highlight the difficulty to significantly impact the latent infection with the currently available drugs. Thus, our results have important implications for the rational design of therapies aimed at reversing HIV latency from diverse cellular reservoirs.

Current Status of Latency Reversing Agents Facing the Heterogeneity of HIV-1 Cellular and Tissue Reservoirs

“…Targeting and reactivating latent cells is challenging due to the heterogeneous nature of the viral reservoirs. Recent studies demonstrating diverse responses of infected cells to LRAs point to their weak effect (Archin et al, 2012;Spivak et al, 2014;Elliott et al, 2015) and highlight the diversity of determinants responsible for the reservoirs' heterogeneity that were demonstrated so far to be virus genetic background- (Norton et al, 2019), cell model- (Spina et al, 2013), cell type- (Baxter et al, 2016;Grau-Expósito et al, 2019;Kula et al, 2019), silencing mechanism- (Elliott et al, 2014;Yukl et al, 2018), tissue reservoir- (Elliott et al, 2014;Telwatte et al, 2018;Yukl et al, 2018), integration site- (Chen et al, 2017;Abner et al, 2018;Battivelli et al, 2018), patient- (Darcis et al, 2017;Yukl et al, 2018), and gender- (Das et al, 2018) specific. In addition, some studies demonstrate a heterogeneous effect of LRAs on NK cells (Garrido et al, 2016) and cytotoxic T-cell lymphocyte (Walker-Sperling et al, 2016) activity with conflicting observations, suggesting either an immunosuppressive effect or a reduced impact of LRA activity on cells sensing HIV-1 reactivation (Archin et al, 2012;Jones et al, 2014;Clutton et al, 2016;Walker-Sperling et al, 2016;Desimio et al, 2017Desimio et al, , 2018.…”

Underestimated effect of intragenic HIV-1 DNA methylation on viral transcription in infected individuals

“…Additionally, we did no specific CD4+ T cells selection. The use of PBMCs could potentially mask differential methylation since it is shown that LRAs have cell-type specific effects, indicating cell-type specific epigenetic profiles [62]. Moreover, due to the targeted nature of the methodology, it does not allow to provide information about integration site methylation or replication competence of the analyzed provirus.…”

Latency reversal agents modulate HIV antigen processing and presentation to CD8 T cells

“…The most efficient LRAs usually induce cellular activation and the addition of immunomodulatory agents (IMA) such as Ruxolitinib [74,75] have been tested ex vivo but even the combination of LRA and IMA modulates HIV antigen processing. New LRAs such as those triggering the non-canonical NF-κB pathway [30] or new LRAs combined with inclusion of TLR ligands [24] or PD-1 blockade [25] will be needed to specifically and efficiently reactivate CD4 cell subsets harboring latent proviruses [55,76]. The present study provides a proof of principle that LRAs, current and future more efficacious drugs, should be assessed not only for their capacity to reactivate HIV transcription or to produce HIV particles in ex vivo assays, but also for their impact on antigen processing and their capacity to promote HIV peptide presentation.…”