HIV-1 latency reversal and immune enhancing activity of IL-15 is not influenced by sex hormones.

The role of different biological variables including biological sex, age, and sex hormones in Human immunodeficiency virus (HIV) cure approaches is not well understood. The γc-cytokine IL-15 is a clinically relevant cytokine that promotes immune activation and mediates HIV reactivation from latency. In this work, we examined the interplay that biological sex, age, and sex hormones 17ß-estradiol, progesterone, and testosterone may have on the biological activity of IL-15. We found that IL-15-mediated CD4+ T cell activation was higher in female donors than in male donors. This difference was abrogated at high 17ß-estradiol concentration. Additionally, there was a positive correlation between age and both IL-15-mediated CD8+ T cell activation and IFN-γ production. In a primary cell model of latency, biological sex, age, or sex hormones did not influence the ability of IL-15 to reactivate latent HIV. Finally, 17ß-estradiol did not consistently affect reactivation of translation-competent reservoirs in CD4+ T cells from people living with HIV who are antiretroviral therapy (ART) suppressed. Our study has found that biological sex and age, but not sex hormones, may influence some of the biological activities of IL-15. Understanding how different biological variables may affect HIV cure therapies will help us evaluate current and future clinical trials aimed toward HIV cure in diverse populations.

HIVepsilon-seq-scalable characterization of intact persistent proviral HIV reservoirs in women.

IMPORTANCE: The lack of a reliable method to accurately detect when replication-competent HIV has been cleared is a major challenge in developing a cure. This study introduces a new approach called the HIVepsilon-seq (HIVε-seq) assay, which uses long-read sequencing technology and bioinformatics to scrutinize the HIV genome at the nucleotide level, distinguishing between defective and intact HIV. This study included 30 participants on antiretroviral therapy, including 17 women, and was able to discriminate between defective and genetically intact viruses at the single DNA strand level. The HIVε-seq assay is an improvement over previous methods, as it requires minimal sample, less specialized lab equipment, and offers a shorter turnaround time. The HIVε-seq assay offers a promising new tool for researchers to measure the intact HIV reservoir, advancing efforts towards finding a cure for this devastating disease.

Brain microglia serve as a persistent HIV reservoir despite durable antiretroviral therapy.

Brain microglia (MG) may serve as a human immunodeficiency virus 1 (HIV) reservoir and ignite rebound viremia following cessation of antiretroviral therapy (ART), but they have yet to be proven to harbor replication-competent HIV. Here, we isolated brain myeloid cells (BrMCs) from nonhuman primates and rapid autopsy of people with HIV (PWH) on ART and sought evidence of persistent viral infection. BrMCs predominantly displayed microglial markers, in which up to 99.9% of the BrMCs were TMEM119+ MG. Total and integrated SIV or HIV DNA was detectable in the MG, with low levels of cell-associated viral RNA. Provirus in MG was highly sensitive to epigenetic inhibition. Outgrowth virus from parietal cortex MG in an individual with HIV productively infected both MG and PBMCs. This inducible, replication-competent virus and virus from basal ganglia proviral DNA were closely related but highly divergent from variants in peripheral compartments. Phenotyping studies characterized brain-derived virus as macrophage tropic based on the ability of the virus to infect cells expressing low levels of CD4. The lack of genetic diversity in virus from the brain suggests that this macrophage-tropic lineage quickly colonized brain regions. These data demonstrate that MG harbor replication-competent HIV and serve as a persistent reservoir in the brain.

Impact of Tamoxifen on Vorinostat-Induced Human Immunodeficiency Virus Expression in Women on Antiretroviral Therapy: AIDS Clinical Trials Group A5366, The MOXIE Trial.

BACKGROUND: Biological sex and the estrogen receptor alpha (ESR1) modulate human immunodeficiency virus (HIV) activity. Few women have enrolled in clinical trials of latency reversal agents (LRAs); their effectiveness in women is unknown. We hypothesized that ESR1 antagonism would augment induction of HIV expression by the LRA vorinostat. METHODS: AIDS Clinical Trials Group A5366 enrolled 31 virologically suppressed, postmenopausal women on antiretroviral therapy. Participants were randomized 2:1 to receive tamoxifen (arm A, TAMOX/VOR) or observation (arm B, VOR) for 5 weeks followed by 2 doses of vorinostat. Primary end points were safety and the difference between arms in HIV RNA induction after vorinostat. Secondary analyses included histone 4 acetylation, HIV DNA, and plasma viremia by single copy assay (SCA). RESULTS: No significant adverse events were attributed to study treatments. Tamoxifen did not enhance vorinostat-induced HIV transcription (between-arm ratio, 0.8; 95% confidence interval [CI], .2-2.4). Vorinostat-induced HIV transcription was higher in participants with increases in H4Ac (fold increase, 2.78; 95% CI, 1.34-5.79) vs those 9 who did not (fold increase, 1.04; 95% CI, .25-4.29). HIV DNA and SCA plasma viremia did not substantially change. CONCLUSIONS: Tamoxifen did not augment vorinostat-induced HIV RNA expression in postmenopausal women. The modest latency reversal activity of vorinostat, postmenopausal status, and low level of HIV RNA expression near the limits of quantification limited assessment of the impact of tamoxifen. This study is the first HIV cure trial done exclusively in women and establishes both the feasibility and necessity of investigating novel HIV cure strategies in women living with HIV. CLINICAL TRIALS REGISTRATION: NCT03382834.

Longitudinal Dynamics of Intact HIV Proviral DNA and Outgrowth Virus Frequencies in a Cohort of Individuals Receiving Antiretroviral Therapy.

BACKGROUND: The replication-competent human immunodeficiency virus (HIV) reservoir is the major barrier to cure. The quantitative viral outgrowth assay (QVOA), the gold-standard method to quantify replication-competent HIV, is resource intensive, which limits its application in large clinical trials. The intact proviral DNA assay (IPDA) requires minimal cell input relative to QVOA and quantifies both defective and intact proviral HIV DNA, the latter potentially serving as a surrogate marker for replication-competent provirus. However, there are limited cross-sectional and longitudinal data on the relationship between IPDA and QVOA measurements. METHODS: QVOA and IPDA measurements were performed on 156 resting CD4 T-cell (rCD4) samples from 83 antiretroviral therapy-suppressed HIV-positive participants. Longitudinal QVOA and IPDA measurements were performed on rCD4 from 29 of these participants. RESULTS: Frequencies of intact, defective, and total proviruses were positively associated with frequencies of replication-competent HIV. Longitudinally, decreases in intact proviral frequencies were strikingly similar to that of replication-competent virus in most participants. In contrast, defective proviral DNA frequencies appeared relatively stable over time in most individuals. CONCLUSIONS: Changes in frequencies of IPDA-derived intact proviral DNA and replication-competent HIV measured by QVOA are similar. IPDA is a promising high-throughput approach to estimate changes in the frequency of the replication-competent reservoir.

Impact of Biological Sex on Immune Activation and Frequency of the Latent HIV Reservoir During Suppressive Antiretroviral Therapy.

BACKGROUND: Persistent HIV infection of long-lived resting CD4 T cells, despite antiretroviral therapy (ART), remains a barrier to HIV cure. Women have a more robust type 1 interferon response during HIV infection relative to men, contributing to lower initial plasma viremia. As lower viremia during acute infection is associated with reduced frequency of latent HIV infection, we hypothesized that women on ART would have a lower frequency of latent HIV compared to men. METHODS: ART-suppressed, HIV seropositive women (n = 22) were matched 1:1 to 22 of 39 ART-suppressed men. We also compared the 22 women to all 39 men, adjusting for age and race as covariates. We measured the frequency of latent HIV using the quantitative viral outgrowth assay, the intact proviral DNA assay, and total HIV gag DNA. We also performed activation/exhaustion immunophenotyping on peripheral blood mononuclear cells and quantified interferon-stimulated gene (ISG) expression in CD4 T cells. RESULTS: We did not observe evident sex differences in the frequency of persistent HIV in resting CD4 T cells. Immunophenotyping and CD4 T-cell ISG expression analysis revealed marginal differences across the sexes. CONCLUSIONS: Differences in HIV reservoir frequency and immune activation appear to be small across sexes during long-term suppressive therapy.

Assessing the impact of AGS-004, a dendritic cell-based immunotherapy, and vorinostat on persistent HIV-1 Infection.

Approaches to deplete persistent HIV infection are needed. We investigated the combined impact of the latency reversing agent vorinostat (VOR) and AGS-004, an autologous dendritic cell immunotherapeutic, on the HIV reservoir. HIV+, stably treated participants in whom resting CD4+ T cell-associated HIV RNA (rca-RNA) increased after VOR exposure ex vivo and in vivo received 4 doses of AGS-004 every 3 weeks, followed by VOR every 72 hours for 30 days, and then the cycle repeated. Change in VOR-responsive host gene expression, HIV-specific T cell responses, low-level HIV viremia, rca-RNA, and the frequency of resting CD4+ T-cell infection (RCI) was measured at baseline and after each cycle. No serious treatment-related adverse events were observed among five participants. As predicted, VOR-responsive host genes responded uniformly to VOR dosing. Following cycles of AGS-004 and VOR, rca-RNA decreased significantly in only two participants, with a significant decrease in SCA observed in one of these participants. However, unlike other cohorts dosed with AGS-004, no uniform increase in HIV-specific immune responses following vaccination was observed. Finally, no reproducible decline of RCI, defined as a decrease of >50%, was observed. AGS-004 and VOR were safe and well-tolerated, but no substantial impact on RCI was measured. In contrast to previous clinical data, AGS-004 did not induce HIV-specific immune responses greater than those measured at baseline. More efficacious antiviral immune interventions, perhaps paired with more effective latency reversal, must be developed to clear persistent HIV infection.

HIV-Specific, Ex Vivo Expanded T Cell Therapy: Feasibility, Safety, and Efficacy in ART-Suppressed HIV-Infected Individuals.

Adoptive T cell therapy has had dramatic successes in the treatment of virus-related malignancies and infections following hematopoietic stem cell transplantation. We adapted this method to produce ex vivo expanded HIV-specific T cells (HXTCs), with the long-term goal of using HXTCs as part of strategies to clear persistent HIV infection. In this phase 1 proof-of-concept study (NCT02208167), we administered HXTCs to antiretroviral therapy (ART)-suppressed, HIV-infected participants. Participants received two infusions of 2 × 107 cells/m2 HXTCs at a 2-week interval. Leukapheresis was performed at baseline and 12 weeks post-infusion to measure the frequency of resting cell infection by the quantitative viral outgrowth assay (QVOA). Overall, participants tolerated HXTCs, with only grade 1 adverse events (AEs) related to HXTCs. Two of six participants exhibited a detectable increase in CD8 T cell-mediated antiviral activity following the two infusions in some, but not all, assays. As expected, however, in the absence of a latency reversing agent, no meaningful decline in the frequency of resting CD4 T cell infection was detected. HXTC therapy in ART-suppressed, HIV-infected individuals appears safe and well tolerated, without any clinical signs of immune activation, likely due to the low residual HIV antigen burden present during ART.

Vorinostat Renders the Replication-Competent Latent Reservoir of Human Immunodeficiency Virus (HIV) Vulnerable to Clearance by CD8 T Cells.

Latently human immunodeficiency virus (HIV)-infected cells are transcriptionally quiescent and invisible to clearance by the immune system. To demonstrate that the latency reversing agent vorinostat (VOR) induces a window of vulnerability in the latent HIV reservoir, defined as the triggering of viral antigen production sufficient in quantity and duration to allow for recognition and clearance of persisting infection, we developed a latency clearance assay (LCA). The LCA is a quantitative viral outgrowth assay (QVOA) that includes the addition of immune effectors capable of clearing cells expressing viral antigen. Here we show a reduction in the recovery of replication-competent virus from VOR exposed resting CD4 T cells following addition of immune effectors for a discrete period. TAKE HOME MESSAGE: VOR exposure leads to sufficient production of viral protein on the cell surface, creating a window of vulnerability within this latent reservoir in antiretroviral therapy (ART)-suppressed HIV-infected individuals that allows the clearance of latently infected cells by an array of effector mechanisms.

Proviral Latency, Persistent Human Immunodeficiency Virus Infection, and the Development of Latency Reversing Agents.

Quiescent proviral genomes that persist during human immunodeficiency virus type 1 (HIV-1) infection despite effective antiretroviral therapy (ART) can fuel rebound viremia after ART interruption and is a central obstacle to the cure of HIV infection. The induction of quiescent provirus is the goal of a new class of potential therapeutics, latency reversing agents (LRAs). The discovery, development, and testing of HIV LRAs is a key part of current efforts to develop latency reversal and viral clearance strategies to eradicate established HIV infection. The development of LRAs is burdened by many uncertainties that make drug discovery difficult. The biology of HIV latency is complex and incompletely understood. Potential targets for LRAs are host factors, and the potential toxicities of host-directed therapies in individuals that are otherwise clinically stable may be unacceptable. Assays to measure latency reversal and assess the effectiveness of potential therapeutics are complex and incompletely validated. Despite these obstacles, novel LRAs are under development and beginning to enter combination testing with viral clearance strategies. It is hoped that the steady advances in the development of LRAs now being paired with emerging immunotherapeutics to clear persistently infected cells will soon allow measurable clinical advances toward an HIV cure.

H3K27 Demethylation at the Proviral Promoter Sensitizes Latent HIV to the Effects of Vorinostat in Ex Vivo Cultures of Resting CD4+ T Cells.

UNLABELLED: Histone methyltransferase inhibitors (HMTis) and histone deacetylase inhibitors (HDACis) are reported to synergistically induce the expression of latent human immunodeficiency virus type 1 (HIV-1), but studies have largely been performed with cell lines. As specific and potent HMTis directed at EZH1 (enhancer of zeste 2 Polycomb repressive complex 2 subunit 1)/EZH2 are now in human testing, we wished to rigorously test such an inhibitor in a primary resting T-cell model of HIV latency. We found that GSK343, a potent and selective EZH2/EZH1 inhibitor, reduced trimethylation of histone 3 at lysine 27 (H3K27) of the HIV provirus in resting cells. Remarkably, this epigenetic change was not associated with increased proviral expression in latently infected resting cells. However, following the reduction in H3K27 at the HIV long terminal repeat (LTR), subsequent exposure to the HDACi suberoylanilide hydroxamic acid or vorinostat (VOR) resulted in increases in HIV gag RNA and HIV p24 antigen production that were up to 2.5-fold greater than those induced by VOR alone. Therefore, in primary resting CD4(+) T cells, true mechanistic synergy in the reversal of HIV latency may be achieved by the combination of HMTis and HDACis. Although other cellular effects of EZH2 inhibition may contribute to the sensitization of the HIV LTR to subsequent exposure to VOR, and to increase viral antigen production, this synergistic effect is directly associated with H3K27 demethylation at nucleosome 1 (Nuc-1). Based upon our findings, the combination of HMTis and HDACis should be considered for testing in animal models or clinical trials. IMPORTANCE: Demethylation of H3K27 mediated by the histone methyltransferase inhibitor GSK343 in primary resting T cells is slow, occurring over 96 h, but by itself does not result in a significant upregulation of cell-associated HIV RNA expression or viral antigen production. However, following H3K27 demethylation, latent viral expression within infected primary resting CD4(+) T cells is synergistically increased upon exposure to the histone deacetylase inhibitor vorinostat. Demethylation at H3K27 sensitizes the HIV promoter to the effects of an HDACi and provides a proof-of-concept for the testing of combination epigenetic approaches to disrupt latent HIV infection, a necessary step toward the eradication of HIV infection.

Staphylococcus aureus Infection in Humanized Mice: A New Model to Study Pathogenicity Associated With Human Immune Response.

BACKGROUND: Staphylococcus aureus is a common pathogen among humans worldwide, with an increasing prevalence of multidrug resistance. The understanding of virulence factors inducing pathogenicity is still incomplete, and thus far the transfer of results from animal studies into successful clinical trials has been difficult. METHODS: In this study, we established an S. aureus infection model in mice engrafted with a human immune system, compared it with infected wild-type and nonhumanized mice, and investigated pathogenesis in these models. RESULTS: Staphylococcus aureus infection was aggravated in humanized mice, compared with wild-type or nonengrafted mice. The humanized mice displayed a significantly reduced survival percentage, increased weight loss, and a more-rapid increase in bacterial burden. In addition, S. aureus infection induced T-cell activation, apoptosis, and Fas receptor expression in humanized but not wild-type mice. CONCLUSIONS: Our findings demonstrate the different pathogenetic mechanisms in wild-type and humanized mice and the possible benefit of including humanized mice in future studies involving S. aureus as a prior step to human clinical trials.

HIV-1 infection, response to treatment and establishment of viral latency in a novel humanized T cell-only mouse (TOM) model.

BACKGROUND: The major targets of HIV infection in humans are CD4⁺ T cells. CD4⁺ T cell depletion is a hallmark of AIDS. Previously, the SCID-hu thy/liv model was used to study the effect of HIV on thymopoeisis in vivo. However, these mice did not develop high levels of peripheral T cell reconstitution and required invasive surgery for infection and analysis. Here, we describe a novel variant of this model in which thy/liv implantation results in systemic reconstitution with human T cells in the absence of any other human hematopoietic lineages. RESULTS: NOD/SCID-hu thy/liv and NSG-hu thy/liv mice were created by implanting human fetal thymus and liver tissues under the kidney capsule of either NOD/SCID or NSG mice. In contrast to NOD/SCID-hu thy/liv mice that show little or no human cells in peripheral blood or tissues, substantial systemic human reconstitution occurs in NSG-hu thy/liv. These mice are exclusively reconstituted with human T cells (i.e. T-cell only mice or TOM). Despite substantial levels of human T cells no signs of graft-versus-host disease (GVHD) were noted in these mice over a period of 14 months. TOM are readily infected after parenteral exposure to HIV-1. HIV replication is sustained in peripheral blood at high levels and results in modest reduction of CD4⁺ T cells. HIV-1 replication in TOM responds to daily administration of combination antiretroviral therapy (ART) resulting in strong suppression of virus replication as determined by undetectable viral load in plasma. Latently HIV infected resting CD4⁺ T cells can be isolated from suppressed mice that can be induced to express HIV ex-vivo upon activation demonstrating the establishment of latency in vivo. CONCLUSIONS: NSG-hu thy/liv mice are systemically reconstituted with human T cells. No other human lymphoid lineages are present in these mice (i.e. monocytes/macrophages, B cells and DC are all absent). These T cell only mice do not develop GVHD, are susceptible to HIV-1 infection and can efficiently maintain virus replication. HIV infected TOM undergoing ART harbor latently infected, resting CD4+ T cells.

Expression of latent HIV induced by the potent HDAC inhibitor suberoylanilide hydroxamic acid.

Histone deacetylases (HDACs) act on histones within the nucleosome-bound promoter of human immunodeficiency virus type 1 (HIV-1) to maintain proviral latency. HDAC inhibition leads to promoter expression and the escape of HIV from latency. We evaluated the ability of the potent inhibitor recently licensed for use in oncology, suberoylanilide hydroxamic acid (SAHA; Vorinostat), selective for Class I HDACs, to induce HIV promoter expression in cell lines and virus production from the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients. In J89, a Jurkat T cell line infected with a single HIV genome encoding the enhanced green fluorescence protein (EGFP) within the HIV genome, SAHA induced changes at nucleosome 1 of the HIV promoter in chromatin immunoprecipitation (ChIP) assays in concert with EGFP expression. In the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients clinically achievable exposures to SAHA induced virus outgrowth ex vivo. These results suggest that potent, selective HDAC inhibitors may allow improved targeting of persistent proviral HIV infection, and define parameters for in vivo studies using SAHA.