Selection of epigenetically privileged HIV-1 proviruses during treatment with panobinostat and interferon-α2a.

CD4+ T cells with latent HIV-1 infection persist despite treatment with antiretroviral agents and represent the main barrier to a cure of HIV-1 infection. Pharmacological disruption of viral latency may expose HIV-1-infected cells to host immune activity, but the clinical efficacy of latency-reversing agents for reducing HIV-1 persistence remains to be proven. Here, we show in a randomized-controlled human clinical trial that the histone deacetylase inhibitor panobinostat, when administered in combination with pegylated interferon-α2a, induces a structural transformation of the HIV-1 reservoir cell pool, characterized by a disproportionate overrepresentation of HIV-1 proviruses integrated in ZNF genes and in chromatin regions with reduced H3K27ac marks, the molecular target sites for panobinostat. By contrast, proviruses near H3K27ac marks were actively selected against, likely due to increased susceptibility to panobinostat. These data suggest that latency-reversing treatment can increase the immunological vulnerability of HIV-1 reservoir cells and accelerate the selection of epigenetically privileged HIV-1 proviruses.

Attacking Latent HIV with convertibleCAR-T Cells, a Highly Adaptable Killing Platform.

Current approaches to reducing the latent HIV reservoir entail first reactivating virus-containing cells to become visible to the immune system. A critical second step is killing these cells to reduce reservoir size. Endogenous cytotoxic T-lymphocytes (CTLs) may not be adequate because of cellular exhaustion and the evolution of CTL-resistant viruses. We have designed a universal CAR-T cell platform based on CTLs engineered to bind a variety of broadly neutralizing anti-HIV antibodies. We show that this platform, convertibleCAR-T cells, effectively kills HIV-infected, but not uninfected, CD4 T cells from blood, tonsil, or spleen and only when armed with anti-HIV antibodies. convertibleCAR-T cells also kill within 48 h more than half of the inducible reservoir found in blood of HIV-infected individuals on antiretroviral therapy. The modularity of convertibleCAR-T cell system, which allows multiplexing with several anti-HIV antibodies yielding greater breadth and control, makes it a promising tool for attacking the latent HIV reservoir.

Shocking HIV-1 with immunomodulatory latency reversing agents.

The "shock-and-kill" strategy is one of the most explored HIV-1 cure approaches to eliminate latent virus. This strategy is based on HIV-1 reactivation using latency reversing agents (LRAs) to reactivate latent proviruses (the "shock" phase) and to induce subsequent elimination of the reactivated cells by immune responses or virus-induced cytopathic effects (the "kill" phase). Studies using immunomodulatory LRAs such as blockers of immune checkpoint molecules, toll-like receptor agonists, cytokines and CD8+ T cell depleting antibodies showed promising potential as LRAs inducing directly or indirectly cellular pathways known to control HIV transcription. However, the precise molecular mechanisms by which these immunomodulatory LRAs reverse latency remain incompletely understood. Together with the heterogenous nature of HIV-1 latency, this lack of understanding complicates efforts to develop more efficient and safer cure strategies. Hence, deciphering those mechanisms is pivotal in designing approaches to eliminate latent HIV infection.

HIV Reservoirs and Treatment Strategies toward Curing HIV Infection.

Combination antiretroviral therapy (cART) has significantly improved the prognosis of individuals living with human immunodeficiency virus (HIV). Acquired immunodeficiency syndrome has transformed from a fatal disease to a treatable chronic infection. Currently, effective and safe anti-HIV drugs are available. Although cART can reduce viral production in the body of the patient to below the detection limit, it cannot eliminate the HIV provirus integrated into the host cell genome; hence, the virus will be produced again after cART discontinuation. Therefore, research into a cure (or remission) for HIV has been widely conducted. In this review, we focus on drug development targeting cells latently infected with HIV and assess the progress including our current studies, particularly in terms of the "Shock and Kill", and "Block and Lock" strategies.

Nanosome-Mediated Delivery Of Hdac Inhibitors and Oxygen Molecules for the Transcriptional Reactivation of Latent Hiv-Infected Cd4+ T Cells.

The treatment of human immunodeficiency virus (HIV) infection is notoriously difficult due to the ability of this virus to remain latent in the host's CD4+ T cells. Histone deacetylases (HDACs) interfere with DNA transcription in HIV-infected hosts, resulting in viral latency. Therefore, HDAC inhibitors can be used to activate viral transcription in latently infected cells, after which the virus can be eliminated through a shock-and-kill strategy. Here, a drug delivery system is developed to effectively deliver HDAC inhibitors to latent HIV-infected cells. Given that the efficacy of HDAC inhibitors is reduced under hypoxic conditions, oxygen-containing nanosomes are used as drug carriers. Oxygen-containing nanosomes can improve the efficiency of chemotherapy by delivering essential oxygen to cells. Additionally, their phospholipid bilayer structure makes them uniquely well-suited for drug delivery. In this study, a novel drug delivery system is developed by taking advantage of the oxygen carriers in these oxygen nanosomes, incorporating a multi-drug strategy consisting of HDAC inhibitors and PKA activators, and introducing CXCR4 binding peptides to specifically target CD4+ T cells. Oxygen nanosomes with enhanced targeting capability through the introduction of the CXCR4 binding peptide mitigate drug toxicity and slow down drug release. The observed changes in the expression of p24, a capsid protein of HIV, indirectly confirm that the proposed drug delivery system can effectively induce transcriptional reactivation of HIV in latent HIV-infected cells.

Biological evaluation of a phosphate ester prodrug of 10-methyl-aplog-1, a simplified analog of aplysiatoxin, as a possible latency-reversing agent for HIV reactivation.

10-Methyl-aplog-1 (10MA-1), a simplified analog of aplysiatoxin, exhibits a high binding affinity for protein kinase C (PKC) isozymes with minimal tumor-promoting and pro-inflammatory activities. A recent study suggests that 10MA-1 could reactivate latent human immunodeficiency virus (HIV) in vitro for HIV eradication strategy. However, further in vivo studies were abandoned by a dose limit caused by the minimal water solubility of 10MA-1. To overcome this problem, we synthesized a phosphate ester of 10MA-1, 18-O-phospho-10-methyl-aplog-1 (phos-10MA-1), to improve water solubility for in vivo studies. The solubility, PKC binding affinity, and biological activity of phos-10MA-1 were examined in vitro, and the biological activity was comparable with 10MA-1. The pharmacokinetic studies in vivo were also examined, which suggest that further optimization for improving metabolic stability is required in the future.

Systematic HIV-1 promoter targeting with CRISPR/dCas9-VPR reveals optimal region for activation of the latent provirus.

CRISPR/dCas9-based activation systems (CRISPRa) enable sequence-specific gene activation and are therefore of particular interest for the 'shock and kill' cure approach against HIV-1 infections. This approach aims to activate the latent HIV-1 proviruses in infected cells and subsequently kill these cells. Several CRISPRa systems have been shown to specifically and effectively activate latent HIV-1 when targeted to the HIV-1 5'LTR promoter, making them a promising 'shock' strategy. Here, we aimed to evaluate the dCas9-VPR system for its applicability in reversing HIV-1 latency and identify the optimal gRNA target site in the HIV-1 5'LTR promoter leading to the strongest activation of the provirus with this system. We systematically screened the HIV-1 promoter by selecting 14 specific gRNAs that cover almost half of the HIV-1 promoter from the 3' half of the U3 until the beginning of the R region. Screening in several latently HIV-1 infected cell lines showed that dCas9-VPR leads to a high activation of HIV-1 and that gRNA-V and -VII induce the strongest activation of replication competent latent provirus. This data indicates that the optimal activation region in the HIV-1 promoter for the dCas9-VPR system is located -165 to -106 bp from the transcription start site and that it is consistent with the optimal activation region reported for other CRISPRa systems. Our data demonstrates that the dCas9-VPR system is a powerful tool for HIV-1 activation and could be harnessed for the 'shock and kill' cure approach.

TLR1/2 Agonist Enhances Reversal of HIV-1 Latency and Promotes NK Cell-Induced Suppression of HIV-1-Infected Autologous CD4+ T Cells.

The complete eradication of human immunodeficiency virus type 1 (HIV-1) is blocked by latent reservoirs in CD4+ T cells and myeloid lineage cells. Toll-like receptors (TLRs) can induce the reversal of HIV-1 latency and trigger the innate immune response. To the best of our knowledge, there is little evidence showing the "killing" effect of TLR1/2 agonists but only a small "shock" potential. To identify a new approach for eradicating the HIV latent reservoir, we evaluated the effectiveness of SMU-Z1, a novel small-molecule TLR1/2 agonist, in the "shock-and-kill" strategy. The results showed that SMU-Z1 could enhance latent HIV-1 transcription not only ex vivo in peripheral blood mononuclear cells from aviremic HIV-1-infected donors receiving combined antiretroviral therapy but also in vitro in cells of myeloid-monocytic origin targeting the NF-κB and mitogen-activated protein kinase pathways. Interestingly, the activation marker CD69 was significantly upregulated in natural killer (NK) cells, B cells, and monocytes 48 h after SMU-Z1 treatment. Furthermore, SMU-Z1 was able to activate T cells without global T cell activation, as well as increasing NK cell degranulation and gamma interferon (IFN-γ) production, which further block HIV-1-infected CD4+ lymphocytes. In summary, the present study found that SMU-Z1 can both enhance HIV-1 transcription and promote NK cell-mediated inhibition of HIV-1-infected autologous CD4+ T cells. These findings indicate that the novel TLR1/2 agonist SMU-Z1 is a promising latency-reversing agent (LRA) for eradication of HIV-1 reservoirs. IMPORTANCE Multiple in vivo studies showed that many LRAs used in the shock-and-kill approach could activate viral transcription but could not induce killing effectively. Therefore, a dual-function LRA is needed for elimination of HIV-1 reservoirs. We previously developed a small-molecule TLR1/2 agonist, SMU-Z1, and demonstrated that it could upregulate NK cells and CD8+ T cells with immune adjuvant and antitumor properties in vivo. In the present study, SMU-Z1 could activate innate immune cells without global T cell activation, induce production of proinflammatory and antiviral cytokines, and enhance the cytotoxic function of NK cells. We showed that SMU-Z1 displayed dual potential ex vivo in the shock of exposure of latently HIV-1-infected cells and in the kill of clearance of infected cells, which is critical for effective use in combination with therapeutic vaccines or broadly neutralizing antibody treatments aimed at curing AIDS.

The Search for an HIV Cure: Where Do We Go From Here?

Since the first case of an HIV sterilizing cure was published, remarkable progress has been made in our understanding of the mechanisms behind HIV persistence. However, our goal of achieving a safe and broadly-available treatment for sustained HIV remission has proven elusive. In this supplement, we provide a series of articles reviewing the technical hurdles facing the field, key assays to measure HIV persistence and the next-generation of therapeutics for HIV remission.

Nonhuman Primate Testing of the Impact of Different Regulatory T Cell Depletion Strategies on Reactivation and Clearance of Latent Simian Immunodeficiency Virus.

Regulatory T cells (Tregs) may be key contributors to the HIV/SIV latent reservoir, since they harbor high levels of HIV/SIV; reverse CD4+ T cell immune activation status, increasing the pool of resting CD4+ T cells; and impair CD8+ T cell function, favoring HIV persistence. We tested the hypothesis that Treg depletion is a valid intervention toward an HIV cure by depleted Tregs in 14 rhesus macaque (RM) controllers infected with SIVsab, the virus that naturally infects sabaeus monkeys, through different strategies: administration of an anti-CCR4 immunotoxin, two doses of an anti-CD25 immunotoxin (interleukin-2 with diphtheria toxin [IL-2-DT]), or two combinations of both. All of these treatments resulted in significant depletion of the circulating Tregs (>70%) and their partial depletion in the gut (25%) and lymph nodes (>50%). The fractions of CD4+ T cells expressing Ki -67 increased up to 80% in experiments containing IL-2-DT and only 30% in anti-CCR4-treated RMs, paralleled by increases in the inflammatory cytokines. In the absence of ART, plasma virus rebounded to 103 vRNA copies/ml by day 10 after IL-2-DT administration. A large but transient boost of the SIV-specific CD8+ T cell responses occurred in IL-2-DT-treated RMs. Such increases were minimal in the RMs receiving anti-CCR4-based regimens. Five RMs received IL-2-DT on ART, but treatment was discontinued because of high toxicity and lymphopenia. As such, while all treatments depleted a significant proportion of Tregs, the side effects in the presence of ART prevent their clinical use and call for different Treg depletion approaches. Thus, based on our data, Treg targeting as a strategy for HIV cure cannot be discarded.IMPORTANCE Regulatory T cells (Tregs) can decisively contribute to the establishment and persistence of the HIV reservoir, since they harbor high levels of HIV/SIV, increase the pool of resting CD4+ T cells by reversing their immune activation status, and impair CD8+ T cell function, favoring HIV persistence. We tested multiple Treg depletion strategies and showed that all of them are at least partially successful in depleting Tregs. As such, Treg depletion appears to be a valid intervention toward an HIV cure, reducing the size of the reservoir, reactivating the virus, and boosting cell-mediated immune responses. Yet, when Treg depletion was attempted in ART-suppressed animals, the treatment had to be discontinued due to high toxicity and lymphopenia. Therefore, while Treg targeting as a strategy for HIV cure cannot be discarded, the methodology for Treg depletion has to be revisited.

µ-Lat: A mouse model to evaluate human immunodeficiency virus eradication strategies.

A critical barrier to the development of a human immunodeficiency virus (HIV) cure is the lack of a scalable animal model that enables robust evaluation of eradication approaches prior to testing in humans. We established a humanized mouse model of latent HIV infection by transplanting "J-Lat" cells, Jurkat cells harboring a latent HIV provirus encoding an enhanced green fluorescent protein (GFP) reporter, into irradiated adult NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice. J-Lat cells exhibited successful engraftment in several tissues including spleen, bone barrow, peripheral blood, and lung, in line with the diverse natural tissue tropism of HIV. Administration of tumor necrosis factor (TNF)-α, an established HIV latency reversal agent, significantly induced GFP expression in engrafted cells across tissues, reflecting viral reactivation. These data suggest that our murine latency ("µ-Lat") model enables efficient determination of how effectively viral eradication agents, including latency reversal agents, penetrate, and function in diverse anatomical sites harboring HIV in vivo.

Latency Reversal 2.0: Giving the Immune System a Seat at the Table.

PURPOSE OF REVIEW: For most people living with HIV (PLWH), treatment with effective antiretroviral therapy (ART) results in suppression of viremia below the limit of detection of clinical assays, immune reconstitution, reduced immune activation, avoidance of opportunistic infections, and progression to AIDS. However, ART alone is not curative, and HIV persists in a non-replicating, latent form. In this review, we provide a historical perspective on non-specific latency reversal approaches (LRA 1.0) and summarize recent advances in latency reversal strategies that target specific signaling pathways within CD4+ T cells or other immune cells to induce expression of latent HIV (immune-based latency reversal, or LRA 2.0). RECENT FINDINGS: The HIV reservoir is primarily composed of latently infected CD4+ T cells carrying integrated, replication-competent provirus that can give rise to rebound viremia if ART is stopped. Myeloid lineage cells also contribute to HIV latency in certain tissues; we focus here on CD4+ T cells as a sufficient body of evidence regarding latency reversal in myeloid cells is lacking. The immunomodulatory LRA 2.0 approaches we describe include pattern recognition receptor agonists, immune checkpoint inhibitors, non-canonical NF-kB stimulation, and transient CD8+ lymphocyte depletion, along with promising combination strategies. We highlight recent studies demonstrating robust latency reversal in nonhuman primate models. While significant strides have been made in terms of virus reactivation from latency, initial hopes for latency reversal alone to result in a reduction of infected cells, through viral cytopathic effect or an unboosted immune system, have not been realized and it seems clear that even effective latency reversal strategies will need to be paired with an approach that facilitates immune recognition and clearance of cells containing reactivated virus.

Modeling the Effects of Latency Reversing Drugs During HIV-1 and SIV Brain Infection with Implications for the "Shock and Kill" Strategy.

Combination antiretroviral therapy (cART) has greatly increased life expectancy for human immunodeficiency virus-1 (HIV-1)-infected patients. Even given the remarkable success of cART, the virus persists in many different cells and tissues. The presence of viral reservoirs represents a major obstacle to HIV-1 eradication. These viral reservoirs contain latently infected long-lived cells. The "Shock and Kill" therapeutic strategy aims to reactivate latently infected cells by latency reversing agents (LRAs) and kill these reactivated cells by strategies involving the host immune system. The brain is a natural anatomical reservoir for HIV-1 infection. Brain macrophages, including microglia and perivascular macrophages, display productive HIV-1 infection. A mathematical model was used to analyze the dynamics of latently and productively infected brain macrophages during viral infection and this mathematical model enabled prediction of the effects of LRAs applied to the "Shock and Kill" strategy in the brain. The model was calibrated using reported data from simian immunodeficiency virus (SIV) studies. Our model produces the overarching observation that effective cART can suppress productively infected brain macrophages but leaves a residual latent viral reservoir in brain macrophages. In addition, our model demonstrates that there exists a parameter regime wherein the "Shock and Kill" strategy can be safe and effective for SIV infection in the brain. The results indicate that the "Shock and Kill" strategy can restrict brain viral RNA burden associated with severe neuroinflammation and can lead to the eradication of the latent reservoir of brain macrophages.

Latency Reversing Agents: Kick and Kill of HTLV-1?

Human T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), is a retrovirus, which integrates into the host genome and persistently infects CD4+ T-cells. Virus propagation is stimulated by (1) clonal expansion of infected cells and (2) de novo infection. Viral gene expression is induced by the transactivator protein Tax, which recruits host factors like positive transcription elongation factor b (P-TEFb) to the viral promoter. Since HTLV-1 gene expression is repressed in vivo by viral, cellular, and epigenetic mechanisms in late phases of infection, HTLV-1 avoids an efficient CD8+ cytotoxic T-cell (CTL) response directed against the immunodominant viral Tax antigen. Hence, therapeutic strategies using latency reversing agents (LRAs) sought to transiently activate viral gene expression and antigen presentation of Tax to enhance CTL responses towards HTLV-1, and thus, to expose the latent HTLV-1 reservoir to immune destruction. Here, we review strategies that aimed at enhancing Tax expression and Tax-specific CTL responses to interfere with HTLV-1 latency. Further, we provide an overview of LRAs including (1) histone deacetylase inhibitors (HDACi) and (2) activators of P-TEFb, that have mainly been studied in context of human immunodeficiency virus (HIV), but which may also be powerful in the context of HTLV-1.

Combinations of Histone Deacetylase Inhibitors with Distinct Latency Reversing Agents Variably Affect HIV Reactivation and Susceptibility to NK Cell-Mediated Killing of T Cells That Exit Viral Latency.

The 'shock-and-kill' strategy to purge the latent HIV reservoir relies on latency-reversing agents (LRAs) to reactivate the provirus and subsequent immune-mediated killing of HIV-expressing cells. Yet, clinical trials employing histone deacetylase inhibitors (HDACis; Vorinostat, Romidepsin, Panobinostat) as LRAs failed to reduce the HIV reservoir size, stressing the need for more effective latency reversal strategies, such as 2-LRA combinations, and enhancement of the immune responses. Interestingly, several LRAs are employed to treat cancer because they up-modulate ligands for the NKG2D NK-cell activating receptor on tumor cells. Therefore, using in vitro T cell models of HIV latency and NK cells, we investigated the capacity of HDACis, either alone or combined with a distinct LRA, to potentiate the NKG2D/NKG2D ligands axis. While Bortezomib proteasome inhibitor was toxic for both T and NK cells, the GS-9620 TLR-7 agonist antagonized HIV reactivation and NKG2D ligand expression by HDACis. Conversely, co-administration of the Prostratin PKC agonist attenuated HDACi toxicity and, when combined with Romidepsin, stimulated HIV reactivation and further up-modulated NKG2D ligands on HIV+ T cells and NKG2D on NK cells, ultimately boosting NKG2D-mediated viral suppression by NK cells. These findings disclose limitations of LRA candidates and provide evidence that NK cell suppression of reactivated HIV may be modulated by specific 2-LRA combinations.

Diversity of small molecule HIV-1 latency reversing agents identified in low- and high-throughput small molecule screens.

The latency phenomenon produced by human immunodeficiency virus (HIV-1) prevents viral clearance by current therapies, and consequently development of a cure for HIV-1 disease represents a formidable challenge. Research over the past decade has resulted in identification of small molecules that are capable of exposing HIV-1 latent reservoirs, by reactivation of viral transcription, which is intended to render these infected cells sensitive to elimination by immune defense recognition or apoptosis. Molecules with this capability, known as latency-reversing agents (LRAs) could lead to realization of proposed HIV-1 cure strategies collectively termed "shock and kill," which are intended to eliminate the latently infected population by forced reactivation of virus replication in combination with additional interventions that enhance killing by the immune system or virus-mediated apoptosis. Here, we review efforts to discover novel LRAs via low- and high-throughput small molecule screens, and summarize characteristics and biochemical properties of chemical structures with this activity. We expect this analysis will provide insight toward further research into optimized designs for new classes of more potent LRAs.

Structure-Activity Relationship Analysis of Benzotriazine Analogues as HIV-1 Latency-Reversing Agents.

"Shock and kill" therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in vitro in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity in vivo A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.

Novel Latency Reversal Agents for HIV-1 Cure.

Antiretroviral therapy (ART) has rendered HIV-1 infection a treatable illness; however, ART is not curative owing to the persistence of replication-competent, latent proviruses in long-lived resting T cells. Strategies that target these latently infected cells and allow immune recognition and clearance of this reservoir will be necessary to eradicate HIV-1 in infected individuals. This review describes current pharmacologic approaches to reactivate the latent reservoir so that infected cells can be recognized and targeted, with the ultimate goal of achieving an HIV-1 cure.

Alternate NF-κB-Independent Signaling Reactivation of Latent HIV-1 Provirus.

Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 from infected individuals because of the establishment of proviral latency in long-lived cellular reservoirs. The shock-and-kill approach aims to reactivate viral replication from the latent state (shock) using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells (kill) by specific therapeutics. The NF-κB RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat (LTR). Nevertheless, prolonged NF-κB activation contributes to the development of various autoimmune, inflammatory, and malignant disorders. In the present study, we established a cellular model of HIV-1 latency in J-Lat CD4+ T cells that stably expressed the NF-κB superrepressor IκB-α 2NΔ4 and demonstrate that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-κB activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors or specific short hairpin RNAs, c-Jun was identified to be an essential factor binding to the LTR enhancer κB sites and mediating the combined synergistic reactivation effect. Furthermore, acetylsalicylic acid (ASA), a potent inhibitor of the NF-κB activator kinase IκB kinase ß (IKK-ß), did not significantly diminish reactivation in a primary CD4+ T central memory (TCM) cell latency model. The present work demonstrates that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-κB, with the potential to avoid unwanted autoimmune- and or inflammation-related side effects associated with latency-reversing strategies.IMPORTANCE The shock-and-kill approach consists of the reactivation of HIV-1 replication from latency using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-κB is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-κB in HIV-1-infected patients resulting from the combined administration of different LRAs could represent a potential risk, especially in the case of a prolonged treatment. We demonstrate here that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivate HIV-1 from latency, even under conditions where NF-κB activation is repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-κB in patients under combination antiretroviral therapy during the shock-and-kill approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs.

Activation of Latent HIV-1 T Cell Reservoirs with a Combination of Innate Immune and Epigenetic Regulators.

The presence of T cell reservoirs in which human immunodeficiency virus (HIV) establishes latency by integrating into the host genome represents a major obstacle to an HIV cure and has prompted the development of strategies aimed at the eradication of HIV from latently infected cells. The "shock-and-kill" strategy is one of the most pursued approaches to the elimination of viral reservoirs. Although several latency-reversing agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. In this study, we evaluated a novel immune system-mediated approach to clearing the HIV reservoir, based on a combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP (cyclic GMP-AMP) and the FDA-approved histone deacetylase inhibitor resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro Reductions in the proportion of HIV-harboring cells and the total amount of HIV DNA were also observed in CD4+ central memory T (TCM) cells, a primary cell model of latency, where resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cell-associated HIV RNA were detected ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from individuals on suppressive antiretroviral therapy (ART). Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a promising step toward HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation for reducing the viral reservoir and inducing specific death of HIV-infected cells.IMPORTANCE One of the challenges associated with HIV-1 infection is that despite antiretroviral therapies that reduce HIV-1 loads to undetectable levels, proviral DNA remains dormant in a subpopulation of T lymphocytes. Numerous strategies to clear residual virus by reactivating latent virus and eliminating the reservoir of HIV-1 (so-called "shock-and-kill" strategies) have been proposed. In the present study, we use a combination of small molecules that activate the cGAS-STING antiviral innate immune response (the di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV-infected T cell killing in cell lines, primary T lymphocytes, and patient samples. These studies represent a novel strategy for HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells.