Elena Martinelli

Antiretroviral therapy (ART) suppresses HIV replication but does not eliminate persistent viral reservoirs, which can rapidly reseed viremia following treatment interruption. We previously showed that blockade of transforming growth factor beta (TGF-β) signaling in vivo promotes SIV latency reversal and is associated with reversal of cellular metabolic quiescence. Here, using the provirally infected promyelocytic OM10.1 cell line, we investigated the cell-intrinsic mechanisms through which TGF-β regulates HIV reactivation. TGF-β inhibited proviral reactivation following stimulation with distinct latency-reversing agents, and this effect was reversed by pharmacologic inhibition of TGF-β signaling. TGF-β reduced HIV transcription without suppressing TNF-α-induced NF-κB activation, suggesting inhibition through an alternative mechanism. Bulk RNA sequencing revealed broad suppression of c-Myc-associated pathways, oxidative phosphorylation, and mTORC1 signaling. Functional metabolic profiling confirmed marked reductions in both glycolytic and mitochondrial ATP production and decreased mitochondrial membrane polarization. Consistent with a functional link between metabolism and viral reactivation, mitochondrial uncoupling strongly inhibited HIV expression. Finally, MYC overexpression completely rescued HIV transcription and partially restored p24 production in TGF-β-treated cells. Together, these findings identify a cell-intrinsic mechanism through which TGF-β restrains HIV reactivation by imposing a metabolically quiescent state, mediated at least in part through suppression of c-Myc.

We previously demonstrated that blocking TGF-β with galunisertib, a safe, orally available small drug, reactivated latent SIV in vivo by shifting T cells toward a transitional effector phenotype. Here, we investigated the mechanisms underlying this effect using single-cell RNA sequencing, metabolic profiling, and high-dimensional spectral flow cytometry of samples from SIV-infected, antiretroviral therapy–treated (ART-treated) macaques before and after galunisertib. To characterize virus-transcribing, infected cells during ART, we developed a novel, sensitive SIV Transcripts Capture Assay (SCAP) that detected 127 SIV-expressing cells within lymph node single-cell transcriptome libraries. Galunisertib drove broad metabolic reprogramming in CD4 ⁺ T cells, with transcriptional upregulation of inflammatory and mitochondrial biosynthesis pathways, confirmed by Seahorse profiling. Metabolomics revealed increased energy metabolites and amino acids and enhanced metabolic flux without proliferation. SIV transcript–positive cells before galunisertib were metabolically quiescent compared with cells without detectable viral transcripts. After galunisertib, virus-expressing cells showed a dramatic metabolic activation, with upregulation of glycolysis, fatty acid metabolism, and TNF-α signaling. High-dimensional flow cytometry demonstrated effects beyond CD4 ⁺ T cells, including fewer tissue-resident memory T cells, but more inflammatory macrophages. In conclusion, SCAP represents a specific tool for characterizing rare SIV-infected cells transcribing virus during ART, and it reveals TGF-β as a key mediator of viral latency in vivo through metabolic suppression.

Cellular metabolism regulates HIV/SIV replication and reservoir establishment, yet how infection and antiretroviral therapy initiation (ARTi) shape CD4⁺ T cell metabolism in vivo remains poorly defined. Using the SIVmac239 macaque model, we integrated single-cell metabolic profiling (MIST), transcriptomics, lipidomics and genome-scale metabolic modeling to characterize their metabolic remodeling. At peak viremia, CD4⁺ T cells exhibited shutdown of de novo fatty-acid (FA) synthesis, reflected by acetyl-CoA carboxylase-1 (ACC1) downregulation, inhibition of lipid-anabolic reactions, and depletion of membrane phospholipids. This state was driven by type I interferon (IFN-I) responses, and IFN-I suppressed ACC1 in vitro . Pharmacologic inhibition of FA synthesis enhanced T cell activation and exerted direct antiviral effects. Following ARTi, most metabolic pathways in CD4 ⁺ T cells were suppressed, whereas oxidative phosphorylation (OXPHOS) remained elevated and its levels in effector memory cells correlated with cell-associated-vDNA These findings identify IFN-driven FA synthesis suppression as a novel effector mechanism during acute viral infection, and persistent OXPHOS in effector CD4⁺ T cells as a metabolic correlate of early reservoir establishment.

Despite effective suppression of viral replication by antiretroviral therapy (ART), chronic HIV infection remains characterized by persistent low-level inflammation and progressive tissue damage, contributing to premature aging and an array of comorbidities including cardiovascular disease, HIV-associated neurocognitive disorders, liver disease, and fibrosis of multiple organs. Increased levels of transforming growth factor beta (TGF-β), characteristic of chronic HIV infection even in the context of ART, appear to be a common thread explaining these disparate comorbidities. As a pleiotropic cytokine with both immunosuppressive and pro-fibrotic properties, TGF-β exerts complex and sometimes paradoxical effects on the HIV lifecycle and pathogenesis. This review explores the multifaceted roles of TGF-β in HIV infection, with particular focus on three critical areas: immunosuppression, tissue fibrosis, and the regulation of viral latency. We discuss recent advancements in understanding the often-paradoxical role of TGF-β on HIV replication and latency dynamics, and how its different effects contribute to multiple mechanisms underlying HIV persistence, from inhibited immune responses and enhanced viral latency to impaired immune reconstitution. A more comprehensive understanding of the mechanisms by which TGF-β contributes to HIV persistence may illuminate novel therapeutic strategies targeting TGF-β signaling pathways for improved HIV treatment and progression toward functional cure.

Problem: Antiretroviral therapy (ART) during pregnancy is essential to prevent vertical HIV transmission and preserve the health of the mother and child. However, ART in pregnancy has been associated with adverse birth outcomes linked to poor placental development. Immune dysregulation of placental development is an important factor in the development of preeclampsia (PE), a common hypertension disorder of pregnancy. Some studies found an association between ART use at conception or during the first trimester and PE. However, little is known regarding the impact of timing of ART initiation on the immune environment in pregnancy. Methods: To investigate the immune environment in pregnant persons with HIV (PPWH) on ART at conception ( N = 40) compared to PPWH that started ART in the second trimester ( N = 40) we analyzed specimens from the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Perinatal Core Protocol, P1025, concluded in 2013. Results: No difference was found in soluble factors in circulation including PlGF and sFLt‐1, associated with PE. However, upon analysis of PBMC by high dimension flow cytometry, we detected a lower frequency of circulating CD49a ⁺ NK cells, associated with uterine tissue and pregnancy, in PPWH on ART at conception compared with PPWH who started ART in the second trimester. Moreover, PBMC from PPWH on ART at conception expressed higher levels of SERPINB2 in transcriptomics analyses. Conclusions: Our findings shed new insights into the potential impact of ART at conception and suggest the persistence of a dysregulated inflammatory environment compared to PPWH starting ART after the conclusion of placental development.

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirm the latency reversal properties of in vivo TGF-β blockade, decrease viral reservoirs and stimulate immune responses. Treatment of eight female, SIV-infected macaques on ART with four 2-weeks cycles of galunisertib leads to viral reactivation as indicated by plasma viral load and immunoPET/CT with a ⁶⁴Cu-DOTA-F(ab’)₂-p7D3-probe. Post-galunisertib, lymph nodes, gut and PBMC exhibit lower cell-associated (CA-)SIV DNA and lower intact pro-virus (PBMC). Galunisertib does not lead to systemic increase in inflammatory cytokines. High-dimensional cytometry, bulk, and single-cell (sc)RNAseq reveal a galunisertib-driven shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrate that galunisertib, a clinical stage TGF-β inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity.

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of the anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirmed the latency reversal properties of in vivo TGF-β blockade, decreased viral reservoirs and stimulated immune responses. Eight SIV-infected macaques on ART were treated with four 2-week cycles of galunisertib. ART was discontinued 3 weeks after the last dose, and macaques euthanized 6 weeks after ART-interruption (ATI). 7 out of 8 macaques rebounded between week 2 and 6 post-ATI. Galunisertib led to viral reactivation as indicated by plasma viral load and immunoPET/CT with ⁶⁴ Cu-DOTA-F(ab’) ₂ -p7D3-probe. A decrease in cell-associated (CA-)SIV DNA was detected in lymph nodes, gut and PBMC, while intact pro-virus in PBMC decreased by 3-fold. No systemic increase in inflammatory cytokines was observed. High-dimensions cytometry, bulk, and single-cell (sc)RNAseq revealed a shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrated that galunisertib, a clinical stage TGF-β inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity. One-sentence summary: TGF-β blockade drives an effector phenotype in immune cells leading to SIV latency reversal and enhanced immune responses in vivo.

Herpes simplex virus type 2 (HSV-2) is common globally and contributes significantly to the risk of acquiring HIV-1, yet these two sexually transmitted infections (STIs) have not been sufficiently characterized for sexual and gender minorities (SGM) across sub-Saharan Africa. To help fill this gap, we performed a retrospective study using plasma and serum samples from 183 SGM enrolled at the Lagos site of the TRUST/RV368 cohort in Nigeria, assayed them for HSV-2 antibodies with the Kalon ELISA and plasma cytokines and chemokines with Luminex, and correlated the findings with HIV-1 viral loads and CD4 counts. We found an overall HSV-2 prevalence of 36.6% (49.5% and 23.9% among SGM with and without HIV-1, respectively, p<0.001). Moreover, HSV-2 positive status was associated with high circulating concentrations of CCL11 among antiretroviral therapy (ART) treated (p=0.031) and untreated (p=0.015) participants, and with high concentrations of CCL2 in the untreated group (p=0.004), independent of VL. Principal component analysis revealed a strong association of cytokines with HIV-1 viral load independent of HSV-2 status. In conclusion, our study finds that HSV-2 prevalence among SGM with HIV-1 is twice as high than HSV-2 prevalence among SGM without HIV-1 in Lagos and suggests that this is associated with higher levels of certain systemic cytokines. Additional work is needed to further characterize the relationship between HSV-2 and HIV-1 in SGM and help develop targeted therapies for co-infected individuals.