Sustainable Antiviral Efficacy of Rejuvenated HIV-Specific Cytotoxic T Lymphocytes Generated from Induced Pluripotent Stem Cells.

Persistence of HIV latently infected cells is a barrier to HIV cure. The "kick and kill" strategy for a cure includes clearance of the viral reservoir by HIV-specific cytotoxic T lymphocytes (CTLs). However, exhaustion and senescence of T cells accelerates during HIV infection, and does not fully recover, despite complete viral suppression under antiretroviral therapy. We previously established an induced pluripotent stem cell (iPSC) from a parental HIV-specific CTL clone and generated an iPSC-derived rejuvenated HIV-specific CTL clone (iPSC-CTL), which exhibited an early memory phenotype, high proliferation capacity and effector functions in vitro. Here, we assessed the antiviral efficacy of the HIV-specific iPSC-CTL by single- and multiple-round viral suppression assays (VSAs). The HIV-specific iPSC-CTL suppressed viral replication in an HLA-dependent manner with equivalent efficacy to the parental CTL clone in single-round VSA. In multiple-round VSA, however, the ability of the iPSC-CTL to suppress viral replication was longer than that of the parental CTL clone. These results indicate that HIV-specific iPSC-CTL can sustainably exert suppressive pressure on viral replication, suggesting a novel approach to facilitate clearance of the HIV reservoir via adoptive transfer of rejuvenated CTLs. IMPORTANCE Elimination of latently HIV-infected cells is required for HIV cure. In the "kick and kill" strategy proposed for a cure to HIV, the host immune system, including HIV-specific cytotoxic T lymphocytes (CTLs), play a central role in eliminating HIV antigen-expressing cells following reactivation by latency-reversing agents (LRAs). However, CTL dysfunction due to exhaustion and senescence in chronic HIV infection can be an obstacle to this strategy. Adoptive transfer with effective HIV-specific CTLs may be a solution of this problem. We previously generated an induced pluripotent stem cell (iPSC)-derived rejuvenated HIV-specific CTL clone (iPSC-CTL) with high functional and proliferative capacity. The present study demonstrates that iPSC-CTL can survive and suppress HIV replication in vitro longer than the parental CTL clone, indicating the potential of iPSC-CTL to sustainably exert suppressive pressure on viral replication. Adoptive transfer with rejuvenated HIV-specific CTLs in combination with LRAs may be a new intervention strategy for HIV cure/remission.

Selective BCL-XL Antagonists Eliminate Infected Cells from a Primary-Cell Model of HIV Latency but Not from Ex Vivo Reservoirs.

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.

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.

Interleukin-15-Stimulated Natural Killer Cells Clear HIV-1-Infected Cells following Latency Reversal Ex Vivo.

Current efforts toward human immunodeficiency virus (HIV) eradication include approaches to augment immune recognition and elimination of persistently infected cells following latency reversal. Natural killer (NK) cells, the main effectors of the innate immune system, recognize and clear targets using different mechanisms than CD8+ T cells, offering an alternative or complementary approach for HIV clearance strategies. We assessed the impact of interleukin 15 (IL-15) treatment on NK cell function and the potential for stimulated NK cells to clear the HIV reservoir. We measured NK cell receptor expression, antibody-dependent cell-mediated cytotoxicity (ADCC), cytotoxicity, interferon gamma (IFN-γ) production, and antiviral activity in autologous HIV replication systems. All NK cell functions were uniformly improved by IL-15, and, more importantly, IL-15-treated NK cells were able to clear latently HIV-infected cells after exposure to vorinostat, a clinically relevant latency-reversing agent. We also demonstrate that NK cells from HIV-infected individuals aviremic on antiretroviral therapy can be efficiently stimulated with IL-15. Our work opens a promising line of investigation leading to future immunotherapies to clear persistent HIV infection using NK cells.IMPORTANCE In the search for an HIV cure, strategies to enhance immune function to allow recognition and clearance of HIV-infected cells following latency reversal are being evaluated. Natural killer (NK) cells possess characteristics that can be exploited for immunotherapy against persistent HIV infection. We demonstrate that NK cells from HIV-positive donors can be strongly stimulated with IL-15, improving their antiviral and cytotoxic potential and, more importantly, clearing HIV-infected cells after latency reversal with a clinically relevant drug. Our results encourage further investigation to design NK cell-based immunotherapies to achieve HIV eradication.

Secreted factors induced by PKC modulators do not indirectly cause HIV latency reversal.

HIV can establish a long-lived latent infection in cells harboring integrated non-expressing proviruses. Latency reversing agents (LRAs), including protein kinase C (PKC) modulators, can induce expression of latent HIV, thereby reducing the latent reservoir in animal models. However, PKC modulators such as bryostatin-1 also cause cytokine upregulation in peripheral blood mononuclear cells (PBMCs), including cytokines that might independently reverse HIV latency. To determine whether cytokines induced by PKC modulators contribute to latency reversal, primary human PBMCs were treated with bryostatin-1 or the bryostatin analog SUW133, a superior LRA, and supernatant was collected. As anticipated, LRA-treated cell supernatant contained increased levels of cytokines compared to untreated cell supernatant. However, exposure of latently-infected cells with this supernatant did not result in latency reactivation. These results indicate that PKC modulators do not have significant indirect effects on HIV latency reversal in vitro and thus are targeted in their latency reversing ability.

Plasma proteomic profiling identifies CD33 as a marker of HIV control in natural infection and after therapeutic vaccination.

BACKGROUND: Biomarkers predicting the outcome of HIV-1 virus control in natural infection and after therapeutic interventions in HIV-1 cure trials remain poorly defined. The BCN02 trial (NCT02616874), combined a T-cell vaccine with romidepsin (RMD), a cancer-drug that was used to promote HIV-1 latency reversal and which has also been shown to have beneficial effects on neurofunction. We conducted longitudinal plasma proteomics analyses in trial participants to define biomarkers associated with virus control during monitored antiretroviral pause (MAP) and to identify novel therapeutic targets that can improve future cure strategies. METHODS: BCN02 was a phase I, open-label, single-arm clinical trial in early-treated, HIV infected individuals. Longitudinal plasma proteomes were analyzed in 11 BCN02 participants, including 8 participants that showed a rapid HIV-1 plasma rebound during a monitored antiretroviral pause (MAP-NC, 'non-controllers') and 3 that remained off ART with sustained plasma viremia <2000 copies/ml (MAP-C, 'controllers'). Inflammatory and neurological proteomes in plasma were evaluated and integration data analysis (viral and neurocognitive parameters) was performed. Validation studies were conducted in a cohort of untreated HIV-1+ individuals (n = 96) and in vitro viral replication assays using an anti-CD33 antibody were used for functional validation. FINDINGS: Inflammatory plasma proteomes in BCN02 participants showed marked longitudinal alterations. Strong proteome differences were also observed between MAP-C and MAP-NC, including in baseline timepoints. CD33/Siglec-3 was the unique plasma marker with the ability to discriminate between MAPC-C and MAP-NC at all study timepoints and showed positive correlations with viral parameters. Analyses in an untreated cohort of PLWH confirmed the positive correlation between viral parameters and CD33 plasma levels, as well as PBMC gene expression. Finally, adding an anti-CD33 antibody to in vitro virus cultures significantly reduced HIV-1 replication and proviral levels in T cells and macrophages. INTERPRETATION: This study indicates that CD33/Siglec-3 may serve as a predictor of HIV-1 control and as potential therapeutic tool to improve future cure strategies. FUNDING: Spanish Science and Innovation Ministry (SAF2017-89726-R and PID2020-119710RB-I00), NIH (P01-AI131568), European Commission (GA101057548) and a Grifols research agreement.

Human Immunodeficiency Virus-1 Latency Reversal via the Induction of Early Growth Response Protein 1 to Bypass Protein Kinase C Agonist-Associated Immune Activation.

Human Immunodeficiency Virus-1 (HIV) remains a global health challenge due to the latent HIV reservoirs in people living with HIV (PLWH). Dormant yet replication competent HIV harbored in the resting CD4+ T cells cannot be purged by antiretroviral therapy (ART) alone. One approach of HIV cure is the "Kick and Kill" strategy where latency reversal agents (LRAs) have been implemented to disrupt latent HIV, expecting to eradicate HIV reservoirs by viral cytopathic effect or immune-mediated clearance. Protein Kinase C agonists (PKCa), a family of LRAs, have demonstrated the ability to disrupt latent HIV to an extent. However, the toxicity of PKCa remains a concern in vivo. Early growth response protein 1 (EGR1) is a downstream target of PKCa during latency reversal. Here, we show that PKCa induces EGR1 which directly drives Tat-dependent HIV transcription. Resveratrol, a natural phytoalexin found in grapes and various plants, induces Egr1 expression and disrupts latent HIV in several HIV latency models in vitro and in CD4+ T cells isolated from ART-suppressed PLWH ex vivo. In the primary CD4+ T cells, resveratrol does not induce immune activation at the dosage that it reverses latency, indicating that targeting EGR1 may be able to reverse latency and bypass PKCa-induced immune activation.

Insights Into Persistent HIV-1 Infection and Functional Cure: Novel Capabilities and Strategies.

Although HIV-1 replication can be efficiently suppressed to undetectable levels in peripheral blood by combination antiretroviral therapy (cART), lifelong medication is still required in people living with HIV (PLWH). Life expectancies have been extended by cART, but age-related comorbidities have increased which are associated with heavy physiological and economic burdens on PLWH. The obstacle to a functional HIV cure can be ascribed to the formation of latent reservoir establishment at the time of acute infection that persists during cART. Recent studies suggest that some HIV reservoirs are established in the early acute stages of HIV infection within multiple immune cells that are gradually shaped by various host and viral mechanisms and may undergo clonal expansion. Early cART initiation has been shown to reduce the reservoir size in HIV-infected individuals. Memory CD4+ T cell subsets are regarded as the predominant cellular compartment of the HIV reservoir, but monocytes and derivative macrophages or dendritic cells also play a role in the persistent virus infection. HIV latency is regulated at multiple molecular levels in transcriptional and post-transcriptional processes. Epigenetic regulation of the proviral promoter can profoundly regulate the viral transcription. In addition, transcriptional elongation, RNA splicing, and nuclear export pathways are also involved in maintaining HIV latency. Although most proviruses contain large internal deletions, some defective proviruses may induce immune activation by expressing viral proteins or producing replication-defective viral-like particles. In this review article, we discuss the state of the art on mechanisms of virus persistence in the periphery and tissue and summarize interdisciplinary approaches toward a functional HIV cure, including novel capabilities and strategies to measure and eliminate the infected reservoirs and induce immune control.

HIV latency reversal agents: A potential path for functional cure?

Despite the advances in Human Immunodeficiency Virus (HIV) treatment, the cure for all HIV patients still poses a major challenge, which needs to be surpassed in the coming years. Among the strategies pursuing this aim, the 'kick-and-kill' approach, which involves the reactivation and elimination of a latent HIV reservoir that resides in some CD4+ T cells, appears promising. The first step of this approach requires the use of latency reversal agents (LRAs) that induce the reactivation of the latent virus. Although several classes of LRAs have been reported so far, some limitations of these compounds still need to be overcome before their clinical use. The complete exhaustion of the reservoir of latent virus will contribute to promote the second step of this approach, facilitating the elimination of the reactivated HIV. Therefore, potent, safe, and non-toxic LRAs are necessary to promote efficient elimination of the HIV-1 virus from its reservoir. In this review article, we focus on the promising LRAs that have been described in the literature over the past few years, highlighting the advantages and disadvantages of their use in the 'kick and kill' approach, thus opening a new avenue in the development of a potential cure.

No evidence of neuronal damage as measured by neurofilament light chain in a HIV cure study utilising a kick-and-kill approach.

OBJECTIVE: HIV-remission strategies including kick-and-kill could induce viral transcription and immune-activation in the central nervous system, potentially causing neuronal injury. We investigated the impact of kick-and-kill on plasma neurofilament light (NfL), a marker of neuro-axonal injury, in RIVER trial participants commencing antiretroviral treatment (ART) during primary infection and randomly allocated to ART-alone or kick-and-kill (ART + vaccination + vorinostat (ART + V + V)). DESIGN: Sub-study measuring serial plasma NfL concentrations. METHODS: Plasma NfL (using Simoa digital immunoassay), plasma HIV-1 RNA (using single-copy assay) and total HIV-1 DNA (using quantitative polymerase chain reaction in peripheral CD4+ T-cells) were measured at randomisation (following ≥22 weeks ART), week 12 (on final intervention day in ART + V + V) and week 18 post-randomisation. HIV-specific T-cells were quantified by intracellular cytokine staining at randomisation and week 12. Differences in plasma NfL longitudinally and by study arm were analysed using mixed models and Student's t-test. Associations with plasma NfL were assessed using linear regression and rank statistics. RESULTS: At randomisation, 58 male participants had median age 32 years and CD4+ count 696 cells/µL. No significant difference in plasma NfL was seen longitudinally and by study arm, with median plasma NfL (pg/mL) in ART-only vs ART + V + V: 7.4 vs 6.4, p = 0.16 (randomisation), 8.0 vs 6.9, p = 0.22 (week 12) and 7.1 vs 6.8, p = 0.74 (week 18). Plasma NfL did not significantly correlate with plasma HIV-1 RNA and total HIV-1 DNA concentration in peripheral CD4+ T-cells at any timepoint. While higher HIV-specific T-cell responses were seen at week 12 in ART + V + V, there were no significant correlations with plasma NfL. In multivariate analysis, higher plasma NfL was associated with older age, higher CD8+ count and lower body mass index. CONCLUSIONS: Despite evidence of vaccine-induced HIV-specific T-cell responses, we observed no evidence of increased neuro-axonal injury using plasma NfL as a biomarker up to 18 weeks following kick-and-kill, compared with ART-only.

Role of Dendritic Cells in Exposing Latent HIV-1 for the Kill.

The development of effective yet nontoxic strategies to target the latent human immunodeficiency virus-1 (HIV-1) reservoir in antiretroviral therapy (ART)-suppressed individuals poses a critical barrier to a functional cure. The 'kick and kill' approach to HIV eradication entails proviral reactivation during ART, coupled with generation of cytotoxic T lymphocytes (CTLs) or other immune effectors equipped to eliminate exposed infected cells. Pharmacological latency reversal agents (LRAs) that have produced modest reductions in the latent reservoir ex vivo have not impacted levels of proviral DNA in HIV-infected individuals. An optimal cure strategy incorporates methods that facilitate sufficient antigen exposure on reactivated cells following the induction of proviral gene expression, as well as the elimination of infected targets by either polyfunctional HIV-specific CTLs or other immune-based strategies. Although conventional dendritic cells (DCs) have been used extensively for the purpose of inducing antigen-specific CTL responses in HIV-1 clinical trials, their immunotherapeutic potential as cellular LRAs has been largely ignored. In this review, we discuss the challenges associated with current HIV-1 eradication strategies, as well as the unharnessed potential of ex vivo-programmed DCs for both the 'kick and kill' of latent HIV-1.

Therapeutic Targeting of HIV Reservoirs: How to Give T Cells a New Direction.

HIV cannot be cured by current antiretroviral therapy (ART) because it persists in a transcriptionally silent form in long-lived CD4+ cells. Leading efforts to develop a functional cure have prioritized latency reversal to expose infected cells to immune surveillance, coupled with enhancement of the natural cytolytic function of immune effectors, or "kick and kill." The most clinically advanced approach to improving the kill is therapeutic immunization, which aims to augment or re-focus HIV-specific cytolytic T cell responses. However, no vaccine strategy has enabled sustained virological control after ART withdrawal. Novel approaches are needed to overcome the limitations of natural adaptive immune responses, which relate to their specificity, potency, durability, and access to tissue reservoirs. Adoptive T cell therapy to treat HIV infection was first attempted over two decades ago, without success. Since then, progress in the field of cancer immunotherapy, together with recognition of the similarities in tumor microenvironments and HIV reservoirs has reignited interest in the application of T cell therapies to HIV eradication. Advances in engineering of chimeric antigen receptor (CAR)-transduced T cells have led to improved potency, persistence and latterly, resistance to HIV infection. Immune retargeting platforms have incorporated non-neutralizing and broadly neutralizing antibodies to generate Bispecific T cell Engagers (BiTEs) and Dual-Affinity Re-Targeting proteins (DARTs). T cell receptor engineering has enabled the development of the first bispecific Immune-mobilizing monoclonal T Cell receptors Against Viruses (ImmTAV) molecules. Here, we review the potential for these agents to provide a better "kill" and the challenges ahead for clinical development.

A Novel Histone Deacetylase Inhibitor, AR-42, Reactivates HIV-1 from Chronically and Latently Infected CD4+ T-cells.

Human immunodeficiency virus type 1 (HIV-1) latency is a major barrier to a cure of AIDS. Latently infected cells harbor an integrated HIV-1 genome but are not actively producing HIV-1. Histone deacetylase (HDAC) inhibitors, such as vorinostat (SAHA), have been shown to reactivate latent HIV-1. AR-42, a modified HDAC inhibitor, has demonstrated efficacy against malignant melanoma, meningioma, and acute myeloid leukemia and is currently used in clinical trials for non-Hodgkin's lymphoma and multiple myeloma. In this study, we evaluated the ability of AR-42 to reactivate HIV-1 in the two established CD4+ T-cell line models of HIV-1 latency. In HIV-1 chronically infected ACH-2 cells, AR-42-induced histone acetylation was more potent and robust than that of vorinostat. Although AR-42 and vorinostat were equipotent in their ability to reactivate HIV-1, AR-42-induced maximal HIV-1 reactivation was twofold greater than vorinostat in ACH-2 and J-Lat (clone 9.2) cells. These data provide rationale for assessing the efficacy of AR-42-mediated HIV-1 reactivation within primary CD4+ T-cells.

A cure for HIV: is it in sight?

HIV is a devastating disease affecting millions of people worldwide despite the advent of successful antiretroviral therapy (ART). However, ART does not result in a cure and has to be taken for life. Accordingly, researchers are turning towards cure efforts, particularly in the light of two patients whose HIV has been seemingly eradicated. Numerous approaches and strategies have been considered for curing HIV, but no scalable and safe solution has yet been reached. With newly discovered difficulties in measuring the HIV reservoir, the main barrier to a cure, the only true test of cure is to stop ART and see whether the virus becomes detectable. However, it is possible that this treatment interruption may be associated with certain risks for patients. Here, we compare the current major approaches and recent advances for curing HIV, as well as discuss ways of evaluating HIV cure and the safety concerns involved.