Search Results (54)

Persistent HIV-1 reservoirs remain a major barrier to a durable functional cure despite long-term suppressive antiretroviral therapy. Although resting memory CD4⁺ T cells constitute the best-characterized cellular reservoir, tissue microenvironments shape viral persistence and immune clearance. The rectal mucosa represents a specialized tissue niche containing HIV-susceptible target cells, antigen-presenting cells, microbial products, inflammatory cues, and local metabolic signals. Within this setting, myeloid-lineage cells, particularly tissue-resident macrophages and dendritic cells, may contribute to HIV-1 persistence through mechanisms distinct from classical T-cell latency. Here, we review how rectal mucosal macrophages may support HIV-1 persistence through longevity, resistance to apoptosis, metabolic adaptation, epigenetic regulation, and sequestration of virions within virus-containing compartments. We also discuss the dual role of mucosal dendritic cells as sentinels that capture and transfer HIV-1 to CD4⁺ T cells, while considering the limited evidence for inducible proviral persistence in selected anatomical and cellular contexts. Importantly, we further distinguish bona fide reservoir-bearing cells from reservoir-supportive mechanisms, including viral capture, trans-infection, immune suppression, and niche-mediated protection. We also highlight how mucosal dysbiosis, barrier disruption, microbial metabolites, chronic interferon signaling, and immunoregulatory myeloid programs may stabilize HIV-1 persistence in rectal tissues. Integrating intact proviral assays, functional measurements, single-cell profiling, multiplex imaging, and spatial transcriptomics will be critical for defining myeloid-associated persistence and guiding tissue-targeted HIV-1 cure strategies. Full article

Protease inhibitors (PIs) remain important components of HIV-2 treatment, but resistance genotyping is frequently challenging in individuals with low or undetectable plasma viremia. Proviral DNA sequencing may provide access to archived viral variants and improve understanding of long-term resistance and clinical evolution. In this retrospective longitudinal study, 27 individuals with HIV-2, both ART-experienced and ART-naïve, followed at a hospital in Lisbon, were analyzed. The HIV-2 protease gene was amplified from peripheral blood mononuclear cell-derived proviral DNA, cloned, and sequenced (Sanger sequencing) at baseline and, for ART-treated participants, after eight years of follow-up. Resistance profiles were interpreted using the Stanford HIVdb, HIV-2EU, and Rega algorithms. Clinical data, including ART history, CD4 counts, and plasma viral load, were collected longitudinally. Amino acid diversity was assessed using Shannon entropy, and longitudinal CD4 dynamics were evaluated using mixed-effects models with time-varying ART exposure. Sensitivity analyses were performed using generalized estimating equations (GEE). A total of 222 clonal HIV-2 protease sequences clustered within group A. Major PI resistance mutations were detected in 21.4% of ART-experienced and 23.1% of ART-naïve individuals at baseline. Longitudinal resistance trajectories varied across participants, including persistence, apparent emergence, and non-detection of previously identified mutations. Mixed-effects modeling revealed substantial inter-individual variability in CD4 trajectories, with no statistically significant associations observed between CD4 evolution and ART status, time, or their interaction. GEE analyses yielded consistent results, supporting robustness across modeling frameworks. Entropy analysis identified localized sequence diversity changes restricted to a small number of protease residues, with positions 60 and 75 differing between groups at baseline and position 21 showing longitudinal variation among treated participants. This study demonstrates that proviral DNA sequencing captures archived HIV-2 protease diversity and reveals persistent and dynamic resistance patterns within the viral reservoir. While no population-level association between ART exposure and CD4 trajectory was observed, marked inter-individual variability highlights the complexity of longitudinal immune recovery in HIV-2 infection. These findings support the value of proviral sequencing as a complementary research tool for characterizing long-term viral evolution in settings where plasma-based genotyping is limited. Full article

Background/Objectives: As important post-transcriptional and epigenetic regulators of gene expression, miRNAs play a pivotal role in modulating host–virus interactions. While prior reviews have addressed either direct miRNA–HIV genome interactions or miRNA-mediated immune modulation in isolation, the integrated dual functionality of these molecules has not been systematically characterized. This review aimed to comprehensively explore how miRNAs that target the HIV-1 genome simultaneously modulate key innate and adaptive host immune signaling pathways. The conceptual novelty of this study is determined not by the identification of previously unknown miRNA-target gene pairs, but by the systemic integration of two regulatory levels (direct inhibition of the viral genome and modulation of the host cell immune signaling pathways) within a unified analytical framework. Such an integrated approach reveals a proviral regulatory network that remains non-obvious when each of these levels is examined separately. Methods: A narrative review was conducted using PubMed, Scopus, Web of Science, and Google Scholar (all years through 2025). In Stage 1, publications reporting experimentally confirmed interactions between host miRNAs and the HIV-1 genome were identified, yielding a curated set of 15 miRNAs. In Stage 2, target genes for each miRNA were retrieved from miRTarBase, TarBase (experimentally validated) and TargetScan 8.0 (in silico predicted). In Stage 3, target genes were manually mapped to key immune signaling pathways (TLR, NF-κB, JAK-STAT). In Stage 4, targeted literature searches were performed for each miRNA–target gene pair to identify direct experimental evidence of interaction. All stages were performed by two independent researchers, with discrepancies resolved by a third. Results: Fifteen host miRNAs with experimentally confirmed binding to the HIV-1 genome were identified, targeting viral genes including nef, pol, vpr, gag, env, vif, and the 3′-UTR. Thirteen of these miRNAs were found to regulate components of major immune pathways. miR-92a-3p, miR-29a/b-3p, miR-150-5p, and miR-125b-5p emerged as the most pleiotropic regulators, simultaneously suppressing TLR signaling (TLR3, TLR7, TLR8, MyD88, TRAF3/6, IRAK1/4), NF-κB components (REL, RELA, NFKB1), JAK-STAT effectors (STAT1–3, STAT5A/B, JAK2), and negative regulators of cytokine signaling (SOCS and PIAS family proteins). miR-133b and miR-196b-5p were found to selectively regulate SOCS/PIAS proteins without involvement in other analyzed pathways, suggesting potential for selective therapeutic targeting. Conclusions: The analyzed miRNAs exhibit functional dualism, acting as direct post-transcriptional suppressors of the HIV-1 genome while simultaneously functioning as epigenetic modulators of host immune signaling. These two modes of action are not independent but together form a conceptual framework of a self-reinforcing proviral regulatory network that, based on the synthesis of published evidence, is proposed to promote viral latency and immune evasion. The identified miRNAs represent promising, albeit complex, targets for novel therapeutic strategies aimed at eliminating latent HIV reservoirs. Full article

Despite prolonged viral inhibition with combination antiretroviral therapy (ART), HIV-1 survives as genetically intact, replication-capable proviruses within durable CD4+ T-cell fractions, involving central memory, transitional memory, and stem cell-like memory populations, as well as within tissue-resident compartments including lymphoid follicles and gut-associated lymphoid tissue. Reservoir stability is preserved via clonal growth of infected cells and epigenetic processes that impose proviral transcriptional silencing. As a result, current therapeutic approaches seek to either directly alter proviral survival or to improve immune-driven elimination of infected cells. At the molecular level, investigational strategies such as CRISPR–Cas9 and CRISPR–Cas12 gene-editing systems are intended to remove or induce inactivating mutations inside embedded proviral DNA, as well as alter host entrance co-receptors such as CCR5 to provide cellular resistance to infection. In addition, pharmacologic latency regulation is being studied via histone deacetylase inhibitors, protein kinase C agonists, and bromodomain inhibitors to reverse latency, along with Tat inhibitors and other transcriptional repressors aimed to persistently silence proviral expression. Moreover, immunological techniques aim to counteract inefficient endogenous antiviral defenses. Broadly neutralizing antibodies with tailored Fc-driven effector functions are under examination for both neutralization and antibody-dependent cellular cytotoxicity. Therapeutic vaccine approaches seek to elevate polyfunctional HIV-specific CD8+ T-cell responses, while adoptive cellular approaches, involving CAR-T cells aiming HIV envelope epitopes, remain in early clinical research. Immune checkpoint blockade is also being investigated to reverse T-cell depletion inside reservoir-rich tissues. Nevertheless, the key obstacles continue to be the diverse reservoir composition, restricted tissue penetration, viral escape, and safety limitations. The molecular and translational obstacles that characterize attempts toward an HIV cure must be addressed through ongoing multidisciplinary research. Full article

In people taking antiretroviral therapy (ART) for HIV infection, the methods to characterize latent and active HIV reservoirs remain costly and labor-intensive. Our objective was to develop a relatively low-cost technique to amplify and sequence the proviruses that persist during ART along with the site in the human genome where each provirus is integrated. We developed a novel HIV-specific Multiple Displacement Amplification (HIV-MDA) assay that specifically amplifies HIV-1 proviruses and their associated integration site. Upon comparison of our HIV-MDA to an established commercial kit designed to amplify cellular DNA, we found that the HIV-MDA (1) typically yielded a greater number of HIV integration site (HIV IS) sequences per 150,000 cells analyzed; (2) improved rates of proviral DNA amplification; and (3) amplified HIV IS at a fraction of the cost (13.6 times less expensive). Thus, the HIV-MDA method appears to be a more sensitive and cost-effective approach to sequencing HIV IS and the associated proviruses compared to a commercial kit. Full article

The Intact Proviral DNA Assay (IPDA) is widely used to quantify genome-intact HIV proviruses in people living with HIV, but viral sequence diversity has been observed to cause assay failures due to primer/probe mismatches. Adapted for subtype C, IPDA-BC is a modified version of the IPDA validated on South African HIV-1 subtype C. India is also impacted by subtype C, but IPDA performance within-subtype across geographical regions is not well studied. We analyzed Indian (IN) and South African (ZA) subtype C sequences in silico, hypothesizing that IPDA-BC may underperform with IN viruses. Primer/probe binding was predicted using three increasingly stringent nucleotide mismatch criteria, whose sensitivity and specificity were evaluated against experimental IPDA outcomes. Phylogenetic analyses confirmed that IN and ZA subtype C sequences form distinct clusters with significant compartmentalization (p < 0.003). Across criteria, up to 6–10% decreases in primer/probe binding were observed in IN versus ZA, with the env forward primer being the most affected. These criteria showed low sensitivity (18–53%) and variable specificity (67–100%) in predicting experimental outcomes. In conclusion, even within subtype, HIV-1 variation across geographical regions may impact IPDA performance, underscoring the need for improved predictive models to guide assay design for global HIV cure research. Full article

The dynamics of the HIV reservoir during antiretroviral therapy (ART) exhibit variability, with a pronounced decline during the initial years of treatment. However, the identification of biomarkers and host factors associated with the decay of the different forms of HIV proviruses remains to be fully elucidated. We conducted a longitudinal study on people with HIV provided by the Spanish National HIV cohort. We assessed the HIV-DNA levels by Intact Proviral DNA Assay, and inflammatory markers using the Proximity Extension Assay, before and after ART initiation. A multivariate linear regression model was employed to identify potential predictive markers. Our results highlight the identification of novel inflammatory markers, such as ADA, DNER, CDCP1, SCF, among others, that varied significantly over ART initiation. In addition, we observed several markers associated with intact HIV-DNA before ART initiation (CD8A, CX3CL1, and ST1A1) or during undetectable viral load post-ART (IL-10). Moreover, up to five markers were able to predict the intact HIV reservoir decay over ART. The strongest predictor was Stem Cell Factor (SCF), where higher baseline levels of this marker were associated with a greater decline in the intact HIV reservoir. In conclusion, we have identified inflammatory markers associated with the size and dynamics of the HIV-DNA reservoir. These findings provide new insights that could contribute to the development of multi-targeted intervention strategies aimed at modulating or monitoring the HIV reservoir size. Full article

HIV-1 proviral landscapes were investigated using near-full-length HIV single-genome sequencing on blood samples from five children with vertically acquired infection and on ART for ~7–9 years. Proviral structures were compared to published datasets in children prior to ART, children on short-term ART, and adults on ART. We found a strong selection for large internal proviral deletions in children, especially deletions of the env gene. Only 2.5% of the proviruses were sequence-intact, lower than in the comparative datasets from adults. Of the proviruses that retained the env gene, >80% contained two or more defects, most commonly stop codons and/or gag start mutations. Significantly fewer defects in the major splice donor site (MSD) and packaging signal were found in the children on short or long-term ART compared to the adults, and tat was more frequently defective in children. These results suggest that different selection pressures may shape the proviral landscape in children compared to adults and reveal potentially different genetic regions to target for measuring the intact HIV reservoir and for achieving HIV remission in children. Full article

A proviral reservoir persists within the central nervous system (CNS) of people with HIV, but its characteristics remain poorly understood. Research has primarily focused on cerebrospinal fluid (CSF), as acquiring brain tissue is challenging. We examined size, cellular tropism, and infection-dynamics of the viral reservoir in post-mortem brain tissue from five individuals on and off antiretroviral therapy (ART) across three brain regions. Microglia-enriched fractions (CD11b⁺) were isolated and levels of intact proviral DNA were quantified (IPDA). Full-length envelope reporter viruses were generated and characterized in CD4⁺ T cells and monocyte-derived microglia. HIV DNA was observed in microglia-enriched fractions of all individuals, but intact proviruses were identified only in one ART-treated individual, representing 15% of the total proviruses. Phenotypic analyses of clones from this individual showed that 80% replicated efficiently in microglia and CD4⁺ T cells, while the remaining viruses replicated only in CD4⁺ T cells. No region-specific effects were observed. These results indicate a distinct HIV brain reservoir in microglia for all individuals, although intact proviruses were detected in only one. Given the unique immune environment of the CNS, the characteristics of microglia, and the challenges associated with targeting these cells, the CNS reservoir should be considered in cure strategies. Full article

Human Immunodeficiency Virus (HIV) proviral reservoirs are cells that harbor integrated HIV proviral DNA within their nuclear genomes. These cells form a heterogeneous group, represented by peripheral blood mononuclear cells (PBMCs), tissue-resident lymphoid and monocytic cells, and glial cells of the central nervous system. The importance of studying the properties of proviral reservoirs is connected with the inaccessibility of integrated HIV proviral DNA for modern anti-retroviral therapies (ARTs) that block virus reproduction. If treatment is not effective enough or is interrupted, the proviral reservoir can reactivate. Early initiation of ART improves the prognosis of the course of HIV infection, which is explained by the reduction in the proviral reservoir pool observed in the early stages of the disease. Different HIV subtypes present differences in the number of latent reservoirs, as determined by structural and functional differences. Unique signatures of patients with HIV, such as elite controllers, have control over viral replication and can be said to have achieved a functional cure for HIV infection. Uncovering the causes of this phenomenon will bring humanity closer to curing HIV infection, potential approaches to which include stem cell transplantation, clustered regularly interspaced short palindromic repeats (CRISPR)/cas9, “Shock and kill”, “Block and lock”, and the application of broad-spectrum neutralizing antibodies (bNAbs). Full article

Despite its effectiveness in controlling plasma viremia, antiretroviral therapy (ART) cannot target proviral DNA, which remains an obstacle to HIV-1 eradication. When treatment is interrupted, the reservoirs can act as a source of viral rebound, highlighting the value of proviral DNA as an additional source of information on an individual’s overall resistance burden. In cases where the viral load is too low for successful HIV-1 RNA genotyping, HIV-1 DNA can help identify resistance mutations in treated individuals. The absence of treatment history, the need to adjust ART despite undetectable viremia, or the presence of LLV further support the use of genotypic resistance tests (GRTs) on HIV-1 DNA. Conventionally, GRTs have been achieved through Sanger sequencing, but the advances in NGS are leading to an increase in its use, allowing the detection of minority variants present in less than 20% of the viral population. The clinical significance of these mutations remains under debate, with interpretations varying based on context. Additionally, proviral DNA is subject to APOBEC3-induced hypermutation, which can lead to defective, nonviable viral genomes, a factor that must be considered when performing GRTs on HIV-1 DNA. Full article

Background: Gastrointestinal mucosal damage due to human immunodeficiency virus (HIV) infection leads to microbial translocation and immune activation, contributing to the development of non-infectious comorbidities (NICM) in people living with HIV (PLWH). Additionally, persistent proviral HIV-1 in the gut-associated lymphatic tissue (GALT) can trigger immunological changes in the epithelial environment, impacting the mucosal barrier. However, the role of zonulin, a modulator of epithelial tight junctions in GALT during HIV infection, remains poorly understood. Methods: We measured zonulin in serum and intestinal tissue sections from five treatment-naive (HIV⁺_NAIVE) and 10 cART-treated (HIV⁺_cART) HIV⁺ individuals, along with 11 controls (CTRL). We compared zonulin levels with clinical characteristics, inflammatory markers (IFN-α, CXCR3, and PD-1), and the viral reservoir in peripheral blood (PB) and terminal ileum (TI). Results: Upon HIV infection, TI was found to harbor more HIV DNA than PB. Circulating zonulin levels were highest in HIV⁺_NAIVE compared to HIV⁺_cART or CTRL. Surprisingly, in the gut tissue sections, zonulin levels were higher in CTRL than in HIV⁺ individuals. Elevated circulating zonulin levels were found to be correlated with CD4⁺T-cell depletion in PB and TI, and with intestinal IFN-α. Conclusions: The findings of this study indicate a shift in zonulin levels from the gut to the bloodstream in response to HIV infection. Furthermore, elevated systemic zonulin levels are associated with the depletion of intestinal CD4⁺ T cells and increased gut inflammation, suggesting a potential link between systemic zonulin and intestinal damage. Gaining insight into the regulation of gut tight junctions during HIV infection could offer valuable understanding for preventing NICM in PLWH. Full article

The persistence of the latent viral reservoir is the main hurdle to curing HIV-1 infection. SIV infection of non-human primates (NHPs), namely Indian-origin rhesus macaques, is the most relevant and widely used animal model to evaluate therapies that seek to eradicate HIV-1. The utility of a model ultimately rests on how accurately it can recapitulate human disease, and while reservoirs in the NHP model behave quantitatively very similar to those of long-term suppressed persons with HIV-1 (PWH) in the most salient aspects, recent studies have uncovered key nuances at the clonotypic level that differentiate the two in qualitative terms. In this review, we will highlight differences relating to proviral intactness, clonotypic structure, and decay rate during ART between HIV-1 and SIV reservoirs and discuss the relevance of these distinctions in the interpretation of HIV-1 cure strategies. While these, to some degree, may reflect a unique biology of the virus or host, distinctions among the proviral landscape in SIV are likely to be shaped significantly by the condensed timeframe of NHP studies. ART is generally initiated earlier in the disease course, and animals are virologically suppressed for shorter periods before receiving interventions. Because these are experimental variables dictated by the investigator, we offer guidance on study design for cure-related studies performed in the NHP model. Finally, we highlight the case of GS-9620 (Vesatolimod), an antiviral TLR7 agonist tested in multiple independent pre-clinical studies in which virological outcomes may have been influenced by study-related variables. Full article

There is currently no cure for HIV infection although adherence to effective antiretroviral therapy (ART) suppresses replication of the virus in blood, increases CD4⁺ T-cell counts, reverses immunodeficiency, and increases life expectancy. Despite these substantial advances, ART is a lifelong treatment for people with HIV (PWH) and upon cessation or interruption, the virus quickly rebounds in plasma and anatomic sites, including the central nervous system (CNS), resulting in disease progression. With recent advances in quantifying viral burden, detection of genetically intact viral genomes, and isolation of replication-competent virus from brain tissues of PWH receiving ART, it has become apparent that the CNS viral reservoir (largely comprised of macrophage type cells) poses a substantial challenge for HIV cure strategies. Other obstacles impacting the curing of HIV include ageing populations, substance use, comorbidities, limited antiretroviral drug efficacy in CNS cells, and ART-associated neurotoxicity. Herein, we review recent findings, including studies of the proviral integration sites, reservoir decay rates, and new treatment/prevention strategies in the context of the CNS, together with highlighting the next steps for investigations of the CNS as a viral reservoir. Full article

Despite remarkable progress, a cure for HIV-1 infection remains elusive. Rebound competent latent and transcriptionally active reservoir cells persevere despite antiretroviral therapy and rekindle infection due to inefficient proviral silencing. We propose a novel “block-lock-stop” approach, entailing long term durable silencing of viral expression towards an irreversible transcriptionally inactive latent provirus to achieve long term antiretroviral free control of the virus. A graded transformation of remnant HIV-1 in PLWH from persistent into silent to permanently defective proviruses is proposed, emulating and accelerating the natural path that human endogenous retroviruses (HERVs) take over millions of years. This hypothesis was based on research into delineating the mechanisms of HIV-1 latency, lessons from latency reversing agents and advances of Tat inhibitors, as well as expertise in the biology of HERVs. Insights from elite controllers and the availability of advanced genome engineering technologies for the direct excision of remnant virus set the stage for a rapid path to an HIV-1 cure. Full article