HIV Latency Reversal Research Articles

Targeting Ikaros and Aiolos with pomalidomide fails to reactivate or induce apoptosis of the latent HIV reservoir.

HIV persists in people living with HIV (PLHIV) on antiretroviral therapy (ART) in long-lived and proliferating latently infected CD4+ T cells that selectively express pro-survival proteins, including the zinc finger proteins, Ikaros and Aiolos. In this study, we investigated whether pomalidomide, an immunomodulatory agent that induces degradation of Ikaros and Aiolos, could increase the death of HIV-infected cells and/or reverse HIV latency. Using an in vitro model of CD4+ T cells infected with a green fluorescent protein (GFP) reporter virus, pomalidomide increased the expression of the pro-survival protein B cell lymphoma (Bcl)-2 and did not increase apoptosis of GFP+ HIV productively infected CD4+ T cells. Pomalidomide also increased the expression of CD155 and UL16-binding protein (ULBP) stress proteins on GFP+ HIV productively infected CD4+ T cells, but this did not translate to enhanced clearance following co-culture with a natural killer (NK) cell line. Using CD4+ T cells from PLHIV on ART, pomalidomide ex vivo activated memory CD4+ T cells resulting in elevated HLA-DR expression and induced CD4+ T cell proliferation but only in the presence of T cell receptor stimulation with anti-CD3 and anti-CD28. There was no effect on cell-associated HIV RNA or the frequency of intact HIV DNA. In conclusion, despite an increase in stress protein expression, promoting Ikaros and Aiolos degradation in CD4+ T cells using pomalidomide did not directly induce apoptosis of HIV-infected cells or induce HIV latency reversal.IMPORTANCEPeople living with HIV (PLHIV) require lifelong antiretroviral therapy (ART) due to the persistence of latently infected cells. The zinc finger proteins, Ikaros and Aiolos, have recently been implicated in promoting the persistence of latently infected cells. In this study, we investigated the effects of pomalidomide, an immunomodulatory imide drug that induces the degradation of Ikaros and Aiolos, on HIV latency reversal and death of infected cells. Using CD4+ T cells from people living with HIV on suppressive antiretroviral therapy, as well as an in vitro model of productive HIV infection, we found that pomalidomide induced T cell activation and expression of stress proteins but no evidence of latency reversal or selective death of infected cells.

Pan-caspase inhibitors induce secretion of HIV-1 latency reversal agent lymphotoxin-alpha from cytokine-primed NK cells

The persistence of HIV-1 latency reservoirs in CD4+ T cells is a significant obstacle for curing HIV-1. Shock-and-kill strategies, which aim to reactivate latent HIV-1 followed by cytotoxic clearance, have shown limited success in vivo due to insufficient efficacy of latency reversal agents (LRAs) and off-target effects. Natural killer (NK) cells, with their ability to mediate cytotoxicity independent of antigen specificity, offer a promising avenue for enhancing the shock-and-kill approach. Previously, we observed that pan-caspase inhibitors induce NK cells to secrete an LRA in vitro. Here, we aimed to identify this LRA using a targeted proteomic approach. We identified lymphotoxin-α (LTα) as the key LRA secreted by NK cells following pan-caspase inhibitor treatment. LTα was shown to significantly induce HIV-1 LTR promoter activity, a hallmark of viral reactivation. Neutralization of LTα effectively abolished the observed LRA activity, confirming its central role. Moreover, cytokine-primed but not resting human primary NK cells exhibited LRA activity that could be neutralized with LTα neutralizing antibodies. Finally, pan-caspase inhibitor treatment did not decrease the ability of the cytokine-primed NK cells to kill target cells. These findings demonstrate that cytokine-primed NK cells, through LTα secretion, can effectively reactivate latent HIV-1 following pan-caspase inhibitor treatment, without compromising NK cell cytotoxicity. This highlights a potential enhancement strategy utilizing NK cells for shock-and-kill approaches in HIV-1 cure research.

Synthesis and preclinical evaluation of tigilanol tiglate analogs as latency-reversing agents for the eradication of HIV.

Tigilanol tiglate (EBC-46) is a selective modulator of protein kinase C (PKC) isoforms that is Food and Drug Administration (FDA) approved for the treatment of mast cell tumors in canines with up to an 88% cure rate. Recently, it has been FDA approved for the treatment of soft tissue sarcomas in humans. The role of EBC-46 and, especially, its analogs in efforts to eradicate HIV, treat neurological and cardiovascular disorders, or enhance antigen density in antigen-targeted chimeric antigen receptor-T cell and chimeric antigen receptor-natural killer cell immunotherapies has not been reported. Enabled by our previously reported scalable synthesis of EBC-46, we report herein the systematic design, synthesis, and evaluation of EBC-46 analogs, including those inaccessible from the natural source and their PKC affinities, ability to translocate PKC, nuclear factor κB activity, and efficacy in reversing HIV latency in Jurkat-Latency cells. Leading analogs show exceptional PKC affinities, isoform selectivities, and functional activities, serving as promising candidates for therapeutic applications.

A high-throughput, microplate reader-based method to monitor in vitro HIV latency reversal in the absence of flow cytometry.

J-Lat cells are derivatives of the Jurkat CD4+ T cell line that contain a non-infectious, inducible HIV provirus with a GFP tag. While these cells have substantially advanced our understanding of HIV latency, their use by many laboratories in low and middle-income countries is restricted by limited access to flow cytometry. To overcome this barrier, we describe a modified J-Lat assay using a standard microplate reader that detects HIV-GFP expression following treatment with latency-reversing agents (LRAs). We show that HIV reactivation by control LRAs like prostratin and romidepsin is readily detected with dose dependence and with significant correlation and sensitivity to standard flow cytometry. For example, 10μM prostratin induced a 20.1±3.3-fold increase in GFP fluorescence in the microplate reader assay, which corresponded to 64.2±5.0% GFP-positive cells detected by flow cytometery. Similarly, 0.3μM prostratin induced a 1.7±1.2-fold increase compared to 8.7±5.7% GFP-positive cells detected. Using this method, we screen 79 epigenetic modifiers and identify CUDC-101, molibresib, and quisinostat as novel LRAs. This microplate reader-based method offers accessibility to researchers in resource-limited regions to work with J-Lat cells and more actively participate in global HIV cure research efforts.

Reactivation of latent HIV-1 by the glucocorticoid receptor modulator AZD9567.

One key determinant of HIV-1 latency reversal is the activation of the viral long terminal repeat (LTR) by cellular transcription factors such as NF-κB and AP-1. Interestingly, the activity of these two transcription factors can be modulated by glucocorticoid receptors (GRs). Furthermore, the HIV-1 genome contains multiple binding sites for GRs. We therefore hypothesized that glucocorticoids and other GR modulators may influence HIV-1 latency and reactivation. To investigate how GR signaling affects latent HIV-1 reservoirs, we assembled a representative panel of GR modulators including natural steroidal agonists, selective and non-selective GR modulators, and clinically approved GR-modulating drugs. The effects of these compounds on HIV-1 reactivation were assessed using latently HIV-1-infected cell lines and primary cells, as well as reporter assays that monitored GR and LTR activities. We found that AZD9567 (Mizacorat), a non-steroidal partial GR agonist, reactivates latent HIV-1 in both lymphoid and myeloid cell lines and primary CD4+ T cells. Conversely, the GR antagonist mifepristone suppresses HIV-1 LTR-driven gene expression. Mechanistic analyses revealed that AZD9567-mediated reactivation partially depends on both GR and AP-1 binding sites in the LTR. In summary, we, here, identify the GR modulator AZD9567 as novel latency-reversing agent that activates LTR-driven gene expression, which may aid in advancing current shock-and-kill approaches in the treatment of HIV-1 infection.IMPORTANCELatently infected cells of people living with HIV are constantly exposed to fluctuating levels of glucocorticoid hormones such as cortisol. In addition, many HIV-infected individuals regularly take corticosteroids as anti-inflammatory drugs. Although corticosteroids are known to affect the activity of the viral long terminal repeat (LTR) promoter and influence ongoing HIV-1 replication, relatively little is known about the effect of corticosteroid hormones and other glucocorticoid receptor (GR) modulators on latent HIV-1. By systematically comparing natural and synthetic GR modulators, we, here, identify a first first-in-class, oral, partial GR agonist that reactivates latent HIV-1 from different cell types. This drug, AZD9567, was previously tested in clinical trials for rheumatoid arthritis. Mutational analyses shed light on the underlying mode of action and revealed transcription factor binding sites in the HIV-1 LTR that determine responsiveness to AZD9567.

PP1.19 – 00036 Unexpected Redundancy of the TNF/NF-kB Axis in HIV-1 Restriction and Latency Reversal in Primary Human MDM Polarized to M1 Cells

PP1.19 – 00036 Unexpected Redundancy of the TNF/NF-kB Axis in HIV-1 Restriction and Latency Reversal in Primary Human MDM Polarized to M1 Cells

3.3 – 00093 RasGRP1 agonists induce cyclin T1 translation to reverse HIV-1 latency in primary CD4+ T cells

3.3 – 00093 RasGRP1 agonists induce cyclin T1 translation to reverse HIV-1 latency in primary CD4+ T cells

PP3.6 – 00064 Novel synthesized protein kinase C modulators show enhanced HIV latency reversal properties and synergize with a BET bromodomain inhibitor

PP3.6 – 00064 Novel synthesized protein kinase C modulators show enhanced HIV latency reversal properties and synergize with a BET bromodomain inhibitor

PP6.7 – 00070 Blockade of HIV-1 Latency Reversal in CD4+ T Cells from ARTsuppressed PLWH by the HIV-1 Antisense Transcript AST

PP6.7 – 00070 Blockade of HIV-1 Latency Reversal in CD4+ T Cells from ARTsuppressed PLWH by the HIV-1 Antisense Transcript AST

PP2.11 – 00011 The cellular factors BRD4 and HSF1 are critical initiators of P-TEFbdependent HIV-1 latency reversal in primary T cells

PP2.11 – 00011 The cellular factors BRD4 and HSF1 are critical initiators of P-TEFbdependent HIV-1 latency reversal in primary T cells

NSC95397 Is a Novel HIV-1 Latency-Reversing Agent.

The latent viral reservoir represents one of the major barriers to curing HIV-1. Focus on the "kick and kill" (also called "shock and kill") approach, in which virus expression is reactivated, and then cells producing virus are selectively depleted, has led to the discovery of many latency-reversing agents (LRAs) that have furthered our understanding of the mechanisms driving HIV-1 latency and latency reversal. Thus far, individual compounds have yet to be robust enough to work as a therapy, highlighting the importance of identifying new compounds that target novel pathways and synergize with known LRAs. In this study, we identified a promising LRA, NSC95397, from a screen of ~4250 compounds. We validated that NSC95397 reactivates latent viral transcription and protein expression from cells with unique integration events and across different latency models. Co-treating cells with NSC95397 and known LRAs demonstrated that NSC95397 synergizes with different drugs under both standard normoxic and physiological hypoxic conditions. NSC95397 does not globally increase open chromatin, and bulk RNA sequencing revealed that NSC95397 does not greatly increase cellular transcription. Instead, NSC95397 downregulates pathways key to metabolism, cell growth, and DNA repair-highlighting the potential of these pathways in regulating HIV-1 latency. Overall, we identified NSC95397 as a novel LRA that does not largely alter global transcription, shows potential for synergy with known LRAs, and may act through novel pathways not previously recognized for their ability to modulate HIV-1 latency.

MALAT1 is important for facilitating HIV-1 latency reversal in latently infected monocytes

Long non-coding RNAs (lncRNAs) are long RNA transcripts with length >200 nucleotides that do not encode proteins. They play a crucial role in regulating HIV-1 infection, yet their involvement in myeloid cells remains underexplored. Myeloid cells are susceptible to HIV infection and contribute substantially to the latent HIV reservoir. Therefore, disruption of latency within these reservoirs is crucial for achieving a definite cure. In this study, we aimed to ascertain the role of MALAT1 lncRNA in reversal of HIV-1 latency. Latently HIV-infected cell line, U1 was treated with SAHA, followed by qRT-PCR assays to confirm HIV-1 reactivation, and MALAT1 expression was assessed. The in vitro experiments revealed a significant increase in MALAT1 expression following latency reactivation and HIV-1 infection, while its knockdown using siRNA resulted in suppression of HIV transcription, which implies that MALAT1 play a significant role in facilitating the reversal of HIV-1 latency by promoting HIV transcription. Clinical samples were analysed to measure MALAT1 and pro-inflammatory cytokines expression. The elevated MALAT1 expression in treatment-naïve subjects compared to those on ART and HIV-negative controls suggests its association with HIV replication. Moreover, correlation analysis revealed a positive association between MALAT1 expression and pro-inflammatory cytokines, TNF-α and IP-10. To conclude, MALAT1 lncRNA emerged as a crucial facilitator of HIV-1 latency reversal in latently infected monocytes by inducing the expression of pro-inflammatory factors. These findings highlight that MALAT1 could potentially be explored as the therapeutic intervention to reactivate latent cells in monocytes.

Transcriptomic study reveals alteration in the expression of long non-coding RNAs (lncRNAs) during reversal of HIV-1 latency in monocytic cell line.

The presence of latent HIV reservoirs continues to be the biggest obstacle to achieving an HIV cure. Thus, long non-coding RNAs (lncRNAs) may serve as the preferred targets for HIV latency reversal. The goal of the study was to identify prospective lncRNAs for subsequent in vitro molecular and functional characterization. RNA-sequencing was performed in latently HIV-infected monocytic cell line (U1) under stimulated and unstimulated condition using Illumina-HiSeqX platform, followed by its validation using qRT-PCR assay. Gene ontology (GO), KEGG pathway, and co-expression analyses were performed to identify the enriched biological processes and pathways in U1 cells post-stimulation with the latency reversal agent SAHA. A total of 3,576 and 1,467 significantly altered lncRNAs and protein-coding genes respectively, were identified in SAHA-stimulated U1 cells compared to unstimulated ones. The GO and KEGG pathway analyses of the differentially expressed protein-coding genes showed the enrichment of diverse biological processes and pathways respectively, in SAHA-stimulated U1 cells. Co-expression analysis between lncRNAs and protein-coding gene pairs, helped predict potential pathways with which these lncRNAs are associated. Further in vitro validation in HIV-infected monocytes showed that the expression of the top two candidate lncRNAs, LINC01231 and LINC00560, are specific to HIV infection. Transcriptome analysis revealed changes in the expression of numerous lncRNAs and protein-coding genes following stimulation with SAHA. Co-expression analysis identified candidate lncRNAs and their associated biological pathways. However, additional in vitro experimental exploration using gene knockdown strategies is needed to ascertain the specific role of LINC01231 and LINC00560 lncRNAs in latently infected monocytes.

HIV-1 latency reversal agent boosting is not limited by opioid use.

Opioid use may affect the HIV-1 reservoir and its reversal from latency. We studied 47 virally suppressed people with HIV (PWH) and observed that lower concentration of HIV-1 latency reversal agents (LRAs), used with small molecules that did not reverse latency, synergistically increased the magnitude of HIV-1 reactivation ex vivo, regardless of opioid use. This LRA boosting, which combined a second mitochondria-derived activator of caspases mimetic or low-dose PKC agonist with histone deacetylase inhibitors, generated more unspliced HIV-1 transcription than PMA with ionomycin (PMAi), the maximal known HIV-1 reactivator. LRA boosting associated with greater histone acetylation, modulated surface activation-induced markers, and altered T cell production of TNF-α, IL-2, and IFN-γ. HIV-1 reservoirs in PWH contained unspliced and polyadenylated virus mRNA, the ratios of which were greater in resting than total CD4+ T cells and corrected to 1:1 with PMAi exposure. We characterized treated suppressed HIV-1 infection as a period of inefficient, not absent, virus transcription. Multiply spliced HIV-1 transcripts and virion production did not consistently increase with LRA boosting, suggesting the presence of a persistent posttranscriptional block. LRA boosting can be leveraged to probe mechanisms of an effective cellular HIV-1 latency reversal program.

Epitranscriptomic m6A modifications during reactivation of HIV-1 latency in CD4+ T cells.

RNA m6A modification is important for regulating gene expression and innate immune responses to HIV-1 infection. However, the functional significance of m6A modification during HIV-1 latency reactivation is unknown. To address this important question, in this study, we used established cellular models of HIV-1 latency, m6A-specific sequencing at single-base resolution, and functional assays. We demonstrate that HIV-1 latency reversal leads to increased levels of cellular m6A modification, correlates with cellular m6A levels, and is dependent on the catalytic activity of the m6A methyltransferase enzyme. We also identified cellular genes that are differentially m6A-modified during HIV-1 reactivation, as well as the sites of m6A within HIV-1 RNA. Our novel findings point toward a significant role for m6A modification in HIV-1 latency reversal.

Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal.

The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing towards inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo. We also detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 prevents full-length HIV RNA production in primary T cells.

A high-throughput, microplate reader-based method to monitor in vitro HIV latency reversal in the absence of flow cytometry.

J-Lat T-cell lines are important to HIV cure research but require flow cytometryWe describe a method to work with J-Lat cells using a standard microplate readerThis assay can detect control LRAs similar to flow cytometry and discover new LRAsThis assay allows low-resourced laboratories to contribute to HIV cure research.

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal.

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.

Immune checkpoint inhibitors in infectious disease.

Following success in cancer immunotherapy, immune checkpoint blockade is emerging as an exciting potential treatment for some infectious diseases, specifically two chronic viral infections, HIV and hepatitis B. Here, we will discuss the function of immune checkpoints, their role in infectious disease pathology, and the ability of immune checkpoint blockade to reinvigorate the immune response. We focus on blockade of programmed cell death 1 (PD-1) to induce durable immune-mediated control of HIV, given that anti-PD-1 can restore function to exhausted HIV-specific T cells and also reverse HIV latency, a long-lived form of viral infection. We highlight several key studies and future directions of research in relation to anti-PD-1 and HIV persistence from our group, including the impact of immune checkpoint blockade on the establishment (AIDS, 2018, 32, 1491), maintenance (PLoS Pathog, 2016, 12, e1005761; J Infect Dis, 2017, 215, 911; Cell Rep Med, 2022, 3, 100766) and reversal of HIV latency (Nat Commun, 2019, 10, 814; J Immunol, 2020, 204, 1242), enhancement of HIV-specific T cell function (J Immunol, 2022, 208, 54; iScience, 2023, 26, 108165), and investigating the effects of anti-PD-1 and anti-CTLA-4 in vivo in people with HIV on ART with cancer (Sci Transl Med, 2022, 14, eabl3836; AIDS, 2021, 35, 1631; Clin Infect Dis, 2021, 73, e1973). Our future work will focus on the impact of anti-PD-1 in vivo in people with HIV on ART without cancer and potential combinations of anti-PD-1 with other interventions, including therapeutic vaccines or antibodies and less toxic immune checkpoint blockers.

Anti-PD-L1 antibody ASC22 in combination with a histone deacetylase inhibitor chidamide as a “shock and kill” strategy for ART-free virological control: a phase II single-arm study

The combination of ASC22, an anti-PD-L1 antibody potentially enhancing HIV-specific immunity and chidamide, a HIV latency reversal agent, may serve as a strategy for antiretroviral therapy-free virological control for HIV. People living with HIV, having achieved virological suppression, were enrolled to receive ASC22 and chidamide treatment in addition to their antiretroviral therapy. Participants were monitored over 24 weeks to measure changes in viral dynamics and the function of HIV-specific CD8+ T cells (NCT05129189). 15 participants completed the study. At week 8, CA HIV RNA levels showed a significant increase from baseline, and the values returned to baseline after discontinuing ASC22 and chidamide. The total HIV DNA was only transiently increased at week 4 (P = 0.014). In contrast, integrated HIV DNA did not significantly differ from baseline. Increases in the proportions of effector memory CD4+ and CD8+ T cells (TEM) were observed from baseline to week 24 (P = 0.034 and P = 0.002, respectively). The combination treatment did not succeed in enhancing the function of HIV Gag/Pol- specific CD8+ T cells. Nevertheless, at week 8, a negative correlation was identified between the proportions of HIV Gag-specific TEM cells and alterations in integrated DNA in the T cell function improved group (P = 0.042 and P = 0.034, respectively). Nine adverse events were solicited, all of which were graded 1 and resolved spontaneously. The combined treatment of ASC22 and chidamide was demonstrated to be well-tolerated and effective in activating latent HIV reservoirs. Further investigations are warranted in the context of analytic treatment interruption.