The persistence of HIV reservoirs, including latently infected, resting CD4+ T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to m ark CD4+ T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4+ T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV+ or SIV+ subjects, we found that most of the circulating memory CD32+ CD4+ T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a TH2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4+ T cells in peripheral blood or lymphoid tissue; isolated CD32+ resting CD4+ T cells accounted for less than 3% of the total H IV DNA in CD4+ T cells. Cell-associated HIV DNA and RNA loads in CD4+ T cells positively correlated with the frequency of CD32+ CD69+ CD4+ T cells but not with CD32 expression on resting CD4+ T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4+ T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4+ T cells enriched for transcriptionally active HIV after long-term ART.

“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.

Antiretroviral therapy (ART) does not cure HIV-1 infection due to the persistence of proviruses in long-lived resting T cells. Strategies targeting these latently infected cells will be necessary to eradicate HIV-1 in infected individuals. Protein kinase C (PKC) activation is an effective mechanism to reactivate latent proviruses and allows for recognition and clearance of infected cells by the immune system. Several ingenol compounds, naturally occurring PKC agonists, have been described to have potent latency reversal activity. We sought to optimize this activity by synthesizing a library of novel ingenols via esterification of the C-3 hydroxyl group of the ingenol core, which itself is inactive for latency reversal. Newly synthesized ingenol derivatives were evaluated for latency reversal activity, cellular activation, and cytotoxicity alongside commercially available ingenols (ingenol-3,20-dibenzoate, ingenol 3-hexanoate, and ingenol-3-angelate) in HIV latency cell lines and resting CD4 T cells from aviremic participants. Among the synthetic ingenols that we produced, we identified several compounds that demonstrate high efficacy and represent promising leads as latency reversal agents for HIV-1 eradication.

Euphorbia usambarica is a traditional medicine used for gynecologic, endocrine, and urogenital illnesses in East Africa; however, its constituents and bioactivities have not been investigated. A variety of compounds isolated from Euphorbia species have been shown to have activity against latent HIV-1, the major source of HIV-1 persistence despite antiretroviral therapy. We performed bioactivity-guided isolation to identify 15 new diterpenoids (1–9, 14–17, 19, and 20) along with 16 known compounds from E. usambarica with HIV-1 latency reversal activity. Euphordraculoate C (1) exhibits a rare 6/6/3-fused ring system with a 2-methyl-2-cyclopentenone moiety. Usambariphanes A (2) and B (3) display an unusual lactone ring constructed between C-17 and C-2 in the jatrophane structure. 4β-Crotignoid K (14) revealed a 250-fold improvement in latency reversal activity compared to crotignoid K (13), identifying that configuration at the C-4 of tigliane diterpenoids is critical to HIV-1 latency reversal activity. The primary mechanism of the active diterpenoids 12–14 and 21 for the HIV-1 latency reversal activity was activation of PKC, while lignans 26 and 27 that did not increase CD69 expression, suggesting a non-PKC mechanism. Accordingly, natural constituents from E. usambarica have the potential to contribute to the development of HIV-1 eradication strategies.

The mitochondrial antiviral signaling protein (MAVS) is part of the cell’s innate immune mechanism of defense. MAVS mRNA is bicistronic and can give rise to a full length-MAVS and a shorter isoform termed miniMAVS. In response to viral infections, viral RNA can be sensed by the cytosolic RNA sensors retinoic acid-inducible gene I (RIG-I) and/or melanoma differentiation-associated protein 5 (MDA5) and activate NF-κB through interaction with MAVS. MAVS can also sense cellular stress and activate an anti-oxidative stress (AOS) response through the activation of NF-κB. Because NF-κB is a main cellular transcription factor for HIV-1, we wanted to address what role MAVS plays in HIV-1 reactivation from latency in CD4 T cells. Our results indicate that RIG-I agonists required full length-MAVS whereas the AOS response induced by Dynasore through its catechol group can reactivate latent HIV-1 in a MAVS dependent manner through miniMAVS isoform. Furthermore, we uncover that PKC agonists, a class of latency-reversing agents, induce an AOS response in CD4 T cells and require miniMAVS to fully reactivate latent HIV-1. Our results indicate that the AOS response, through miniMAVS, can induce HIV-1 transcription in response to cellular stress and targeting this pathway adds to the repertoire of approaches to reactivate latent HIV-1 in ‘shock-and-kill’ strategies.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that specifically target bacterial metabolites but are also identified as innate-like sensors of viral infection. Individuals with chronic HIV-1 infection have lower numbers of circulating MAIT cells compared with healthy individuals, yet the features of the MAIT TCR repertoire are not well known. We isolated and stimulated human PBMCs from healthy non-HIV-infected donors (HD), HIV-infected progressors on antiretroviral therapy, and HIV-infected elite controllers (EC). We sorted MAIT cells using flow cytometry and used a high-throughput sequencing method with bar coding to link the expression of TCRa, TCRb, and functional genes of interest at the single-cell level. We show differential patterns of MAIT TCR usage among the groups. We observed expansions of certain dominant MAIT clones in HIV-infected individuals upon Escherichia coli stimulation, which was not observed in clones of HD. We also found different patterns of CDR3 amino acid distributions among the three groups. Furthermore, we found blunted expression of phenotypic genes in HIV individuals; most notably, HD mounted a robust IFNG response to stimulation, whereas both HIV-infected progressors and EC did not. In conclusion, our study describes the diverse MAIT TCR repertoire of persons with chronic HIV-1 infection and suggest that MAIT clones of HIV-infected persons may be primed for expansion more than that of noninfected persons. Further studies are needed to examine the functional significance of unique MAIT cell TCR usage in EC. ImmunoHorizons, 2021, 5: 360-369.

IntroductionThough antiretroviral therapy has led to viral suppression and increased quality of life for patients living with HIV-1, strategies to eliminate the HIV-1 latent reservoir are still necessary to eliminate HIV. Latency reversal with superior latency reversal agents (LRAs) such as protein kinase C (PKC) agonists is a promising strategy for unveiling and eliminating the latent HIV-1 reservoir. However, PKC agonists induce T cell activation and deleterious pro- inflammatory cytokine production. Secondary pharmacological agents combined with LRAs have been previously shown to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 viral reactivation. Histone deacetylase inhibitors (HDACi) are also known for inhibiting deleterious pro-inflammatory cytokines in the context of graft-versus-host disease and rheumatoid arthritis in addition to being known to synergize with PKC agonists. In this study we investigated whether HDACi and other epigenetic modifiers could decrease PKC- induced pro-inflammatory cytokines secretion while simultaneously synergizing with the PKC agonists Ingenol-3,20-dibenzoate, to enhance latency reversal.MethodsWe screened an epigenetic modifier library in health donor human peripheral blood mononuclear cells (PBMCs) to identify compounds (‘hits’) that reduced intracellular IL-6 pro-inflammatory cytokine production induced by PKC agonist Ingenol-3,20-dibenzoate. We then further tested reducers of intracellular IL-6 (‘hits’) for their ability to synergize with Ingenol-3,20-dibenzoate in the J-LAT 10.6 model of HIV-1 latency. The most promising epigenetic modifier from both screens, the HDACi Panobinostat, was then further tested for its ability to reduce pro-inflammatory cytokines and synergize with Ingenol-3,20-dibenzoate.ResultsWe show that co-treatment with Ingenol-3,20-dibenzoate and Panobinostat reduces pro-inflammatory cytokines and enhances latency reversal in vitro. Panobinostat suppressed pro-inflammatory cytokine production when combined with Ingenol-3,20- dibenzoate ex vivo when using aviremic patient cells, but antagonized Ingenol-3,20-dibenzoate dependent latency reversal ex vivo.ConclusionThe combination of Panobinostat and Ingenol-3,20-dibenzoate reduces deleterious cytokine production but is not a suitable latency reversal combination therapy.

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