Identification of Novel Immunomodulatory Strategies to Tackle HIV-1 Persistence

Abstract

Antiretroviral therapy (ART) has dramatically improved the life expectancy for people with HIV (PLWH), but it is not curative and must be taken for life. The persistence of HIV in transcriptionally silent but replication-competent latent reservoirs is a major barrier to HIV cure. Thus, strategies targeting the latent HIV reservoir are crucial in achieving HIV cure, as the "shock and kill" strategy using latency reversing agents (LRAs) to induce virus transcription, exposing latent HIV to immune clearance, or the permanent silencing of viral transcription using latency promoting agents (LPAs) in the "block and lock" strategy. _x000D_ Considering the heterogeneity of the latent reservoir comprising cells from distinct lineages, we developed non-clonal cellular models of HIV-1 latency using cell lines from lymphoid or myeloid lineage for identifying new LRAs. We performed a high-throughput screening (HTS) of an anticancer compound library using these models of HIV-1 latency, allowing the identification of LRAs with potent activity across multiple cell lineages. Interestingly, we identified two classes of innate immunomodulating agents as novel LRAs in these models: IκB/IKK inhibitors (IKKis) and JAK inhibitors (JAKi). As previously described for acitretin and TLR agonists, modulation of innate immune stimulation could impact viral latency and contribute to the clearance of the HIV reservoir. _x000D_ In the first chapter of the thesis, we focused on validating the LRA capacity of IKKis in both models of HIV latency. Our results show the latency-reversing activity of IKKis in HIV-1 latently infected lymphoid and myeloid compartments. In particular, selective inhibitors targeting TBK1 and IKKε showed relevant HIV reactivation capacities through a mechanism of NF-κB-induced HIV transcription in vitro and ex vivo. Since HIV-associated immune activation and hyperinflammatory responses persist in PLWH, we also evaluated the IKKis effect on immune cell function, observing that the LRA activity of IKKi did not alter the activation status in CD4+T cells, concomitant with significantly decreased proinflammatory responses._x000D_ Next, in the second chapter of the thesis, we characterized the latency reactivation capacity of JAKi as a potential novel therapeutic strategy for HIV-1 cure. We observed that contrary to the previously described role of JAKi as potent inhibitors of latency reactivation, a subclass of selective JAK2 inhibitors (JAK2i) showed a significant capacity for reversing HIV latency. Notably, the JAK2i fedratinib reversed HIV-1 latency in non-clonal lymphoid and myeloid in vitro models of HIV-1 latency and ex vivo in CD4+ T cells from ART+ PLWH, albeit its function was not dependent on JAK2 expression. To delineate the mechanism of action for JAK2i-induced latency reactivation, we performed whole transcriptomic analysis, identifying a significant upregulation of IRF7 expression despite the blockade of the JAK-STAT pathway and downregulation of proinflammatory cytokines and chemokines. Moreover, IRF7 expression levels positively correlated with HIV latency reactivation capacity of JAK2 inhibitors and other common LRAs. _x000D_ Furthermore, we characterized the latency promoting capacity of the JAK2i pacritinib, demonstrating its potency in blocking LRA-induced reactivation in vitro and in ex vivo-treated primary cells from PLWH. These results were consistent with the relevance of JAK-STAT signalling blockade as a strategy to prevent HIV-1 latency reversal. Collectively, these results represent a promising step towards HIV eradication by demonstrating the potential of innate immune modulation for reducing the viral reservoir through a novel pathway driven by IRF7._x000D_ Overall, our studies highlight the diverse outcomes of modulating immune responses to target HIV persistence in latent reservoirs, either reactivating HIV-1 latency as the TBKi/IKKi and the selective JAK2i fedratinib or epigenetic silencing of the provirus as the JAK2i pacritinib, here described. In addition, the capacity for counteracting chronic immune activation and dysregulated inflammatory responses make immune-modulating agents interesting candidates for combination therapy with ART.

Date of Award	30 Sept 2022
Original language	English
Supervisor	Ester Ballana Guix (Director)