A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.

Background: medication-assisted treatment (MAT) with buprenorphine is now widely prescribed to treat addiction to heroin and other illicit opioids. There is some evidence that illicit opioids enhance HIV-1 replication and accelerate AIDS pathogenesis, but the effect of buprenorphine is unknown. Methods: we obtained peripheral blood mononuclear cells (PBMCs) from healthy volunteers and cultured them in the presence of morphine, buprenorphine, or methadone. We infected the cells with a replication-competent CCR5-tropic HIV-1 reporter virus encoding a secreted nanoluciferase gene, and measured infection by luciferase activity in the supernatants over time. We also surveyed opioid receptor expression in PBMC, genital epithelial cells and other leukocytes by qPCR and western blotting. Reactivation from latency was assessed in J-Lat 11.1 and U1 cell lines. Results: we did not detect expression of classical opioid receptors in leukocytes, but did find nociception/orphanin FQ receptor (NOP) expression in blood and vaginal lymphocytes as well as genital epithelial cells. In PBMCs, we found that at physiological doses, morphine, and methadone had a variable or no effect on HIV infection, but buprenorphine treatment significantly increased HIV-1 infectivity (median: 8.797-fold increase with 20 nM buprenorphine, eight experiments, range: 3.570–691.9, p = 0.0078). Using latently infected cell lines, we did not detect reactivation of latent HIV following treatment with any of the opioid drugs. Conclusions: our results suggest that buprenorphine, in contrast to morphine or methadone, increases the in vitro susceptibility of leukocytes to HIV-1 infection but has no effect on in vitro HIV reactivation. These findings contribute to our understanding how opioids, including those used for MAT, affect HIV infection and reactivation, and can help to inform the choice of MAT for people living with HIV or who are at risk of HIV infection.

In addition to their role in protein translation, tRNAs can be cleaved into shorter, biologically active fragments called tRNA fragments (tRFs). Specific tRFs from spermatocytes can propagate metabolic disorders in second generations of mice. Thus, tRFs in germline cells are a mechanism of epigenetic inheritance. It has also been shown that stress and toxins can cause alterations in tRF patterns. We were therefore interested in whether injecting illicit drugs, a major stressor, impacts tRFs in germline cells. We sequenced RNA from spermatocytes and from semen-derived exosomes from people who inject illicit drugs (PWID) and from non-drug using controls, both groups of unknown fertility status. All PWID injected opioids daily, but most also used other illicit drugs. The tRF cleavage products from Gly-GCC tRNA were markedly different between spermatocytes from PWID compared to controls. Over 90% of reads in controls mapped to shorter Gly-GCC tRFs, while in PWID only 45% did. In contrast, only 4.1% of reads in controls mapped to a longer tRFs versus 45.6% in PWID. The long/short tRF ratio was significantly higher in PWID than controls (0.23 versus 0.16, P = 0.0128). We also report differential expression of a group of small nucleolar RNAs (snoRNAs) in semen-derived exosomes, including, among others, ACA14a, U19, and U3-3. Thus, PWID exhibited an altered cleavage pattern of tRNA-Gly-GCC in spermatocytes and an altered cargo of snoRNAs in semen-derived exosomes. Participants were not exclusively using opioids and were not matched with controls in terms of diet, chronic disease, or other stressors, so our finding are not conclusively linked to opioid use. However, all individuals in the PWID group did inject heroin daily. Our study indicates a potential for opioid injection and/or its associated multi-drug use habits and lifestyle changes to influence epigenetic inheritance.

The barrier to HIV cure is the HIV reservoir, which is composed of latently infected CD4⁺ T cells and myeloid cells that carry stably integrated and replication-competent provirus. The gastrointestinal tract (GIT) contains a substantial part of the HIV reservoir and its immunophysiology could be especially conducive for HIV persistence and reactivation. However, the exact cellular microenvironment and molecular mechanisms that govern the renewal of provirus-harboring cells and proviral reactivation in the GIT remain unclear. In this review, we outline the evidence supporting an overarching hypothesis that interferon activity driven by specialized enterocytes creates a microenvironment that fosters proliferation of latently infected CD4⁺ T cells and sporadic HIV reactivation from these cells. First, we describe unique immunologic features of the gastrointestinal associated lymphoid tissue (GALT), specifically highlighting IFN activity in specialized enterocytes and potential interactions between these cells and neighboring HIV susceptible cells. Then, we will describe dysregulation of IFN signaling in HIV infection and how IFN dysregulation in the GALT may contribute to the persistence and reactivation of the latent HIV reservoir. Finally, we will speculate on the clinical implications of this hypothesis for HIV cure strategies and outline the next steps.

To assess whether biomarkers of systemic inflammation are associated with HIV acquisition or with the timing of ART initiation (“immediate”, at diagnosis, versus “deferred”, at 24 weeks post-diagnosis) in men-who-have-sex-with-men (MSM) and transgender women, we conducted a retrospective study comparing inflammatory biomarkers in participants’ specimens collected before infection and after ≥2 years of effective ART. We measured biomarkers in four longitudinally collected plasma, including two specimens collected from each participant before and two after HIV acquisition and confirmed ART-suppression. Biomarkers were quantified by enzyme-linked immuno-assay or Meso Scale Discovery. When evaluating systematic variation in these markers over time, we found that multiple biomarkers consistently varied across participants’ two pre-infection or two post-ART-suppression specimens. Additionally, we compared changes in biomarkers after vs before HIV acquisition. Across 47 participants, the levels of C-reactive protein (CRP), monocyte chemo-attractant protein-1, tumor necrosis factor-α and interferon gamma-induced protein-10 significantly increased while leptin and lipopolysaccharide binding protein (LBP) significantly decreased following HIV infection. Randomization to deferred-ART initiation was associated with greater increases in CRP and no decrease in LBP. Acquisition of HIV appeared to induce systemic inflammation, with elevation of biomarkers previously associated with infections and cardiovascular disease. Initiation of ART during the early weeks of infection tempered the increase in pro-inflammatory biomarkers compared to delaying ART for ~24 weeks after HIV diagnosis. These findings provide insight into potential mediators by which immediate-ART initiation improves health outcomes, perhaps because immediate-ART limits the size of the HIV reservoir or limits immune dysregulation that in turn trigger systemic inflammation.

Objective: Despite effective antiretroviral therapy (ART), people with HIV (PWH) continue to experience elevated morbidity, potentially driven by persistent immune dysregulation. This study aimed to define transcriptomic heterogeneity in peripheral CD4 ⁺ T cells and identify immune signatures associated with clinical stratification in ART-treated PWH. Design: We analyzed transcriptomic and immunologic (plasma cytokine) data from a well-characterized cohort of ART-suppressed PWH to identify biologically meaningful subgroups. Methods: Bulk RNA sequencing was performed on peripheral CD4 ⁺ T cells from 154 ART-treated PWH. Plasma levels of immune mediators were also quantified. We applied Pairwise Controlled Manifold Approximation (PaCMAP), a novel dimensionality reduction technique, to identify transcriptomic clusters and assessed their associations with clinical and immunological parameters, including CD4:CD8 ratio and cell-associated HIV DNA/RNA. Results: PaCMAP identified 3 distinct transcriptomic clusters among PWH. These clusters were enriched for differential expression of genes regulated by NF-κB, suggesting a role for chronic immune activation and inflammation. While clustering was not associated with HIV reservoir size, there was a modest association with CD4:CD8 ratio, a key marker of immune recovery. In addition, plasma levels of IL-1β, TNF-α, and G-CSF differed across clusters, supporting a link between plasma cytokines and CD4 ⁺ T-cell transcriptomic diversity. Conclusions: Our findings define transcriptomic subgroups in ART-treated PWH that are characterized by NF-κB–driven gene expression and distinct inflammatory cytokine profiles. These immune signatures may serve as biomarkers for immunological stratification and provide insights into persistent immune dysfunction despite viral suppression.