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Low-level viremia (LLV) ranging from 50 to 1,000 copies/ml is common in most HIV-1-infected patients receiving antiretroviral therapy (ART). However, the source of LLV and the impact of LLV on the HIV-1 reservoir during ART remain uncertain. We hypothesized that LLV may arise from the HIV reservoir and its occurrence affect the composition of the reservoir after LLV episodes. Accordingly, we investigated the genetic linkage of sequences obtained from plasma at LLV and pre-ART time points and from peripheral blood mononuclear cells (PBMCs) at pre-ART, pre-LLV, LLV, and post-LLV time points. We found that LLV sequences were populated with a predominant viral quasispecies that accounted for 67.29%-100% of all sequences. Two episodes of LLV in subject 1, spaced 6 months apart, appeared to have originated from the stochastic reactivation of latently HIV-1-infected cells. Moreover, 3.77% of pre-ART plasma sequences were identical to 67.29% of LLV-3 plasma sequences in subject 1, suggesting that LLV may have arisen from a subset of cells that were infected before ART was initiated. No direct evidence of sequence linkage was found between LLV viruses and circulating cellular reservoirs in all subjects. The reservoir size, diversity, and divergence of the PBMC DNA did not differ significantly between the pre- and post-LLV sampling points (P > 0.05), but the composition of viral reservoir quasispecies shifted markedly before and after LLV episodes. Indeed, subjects with LLV had a higher total PBMC DNA level, greater viral diversity, a lower proportion of variants with identical sequences detected at two or more time points, and a shorter variant duration during ART compared with subjects without LLV. Overall, our findings suggested that LLV viruses may stem from an unidentified source other than circulating cellular reservoirs. LLV episodes may introduce great complexity into the HIV reservoir, which brings challenges to the development of treatment strategies. [ABSTRACT FROM AUTHOR]

Summary: Background: Among people living with HIV-1 (PHIV), immunological non-responders (INR) experience incomplete immune recovery despite suppressive antiretroviral treatment (ART), facing more severe non-AIDS events than immunological responders (IR) due to higher chronic immune activation and inflammation (cIA/I). We analyzed the HIV-1 reservoir and immunometabolism in monocytes as a source of cIA/I. Methods: Cross-sectional study in which 110 participants were enrolled: 25 treatment-naïve; 35 INR; 40 IR; and 10 healthy controls. Cell-associated HIV-1-DNA (HIV-DNA) and -RNA (HIV-RNA) were measured in FACS-isolated monocytes using digital droplet PCR. Intact, 5′ deleted, and 3′ deleted proviruses were quantified by the intact proviral DNA assay. Systemic inflammation, monocyte immunophenotype, and immunometabolism were characterized by immunoassays, flow cytometry, and real-time cellular bioenergetics measurements, respectively. Findings: Monocytes from INR harbor higher HIV-RNA and HIV-DNA levels than IR. HIV-RNA was found in 14/21 treatment-naïve [2512 copies/106 TBP (331–4666)], 17/33 INR [240 (148–589)], and 15/28 IR [144 (15–309)], correlating directly with sCD163, IP-10, GLUT1high cells and glucose uptake, and inversely with the CD4+/CD8+ ratio. HIV-DNA was identified in all participants with detectable HIV-RNA, with intact provirus in 9/12 treatment-naïve [13 copies/106 monocytes (7–44)], 8/14 INR [46 (18–67)], and 9/13 IR [9 (7–24)]. INR presented glucose metabolism alterations and mitochondrial impairment; decreased coupling efficiency and BHI, and increased mitochondrial dysfunction inversely correlating with the CD4+/CD8+ ratio. Interpretation: HIV-RNA, more than HIV-DNA, in monocytes and their altered metabolism are factors associated with the higher cIA/I that characterize INR. Funding: This work was supported by the European Regional Development Fund, ISCIII, grant PI20/01646. Other funding sources: Instituto de Salud Carlos III through the Subprogram Miguel Servet (CP19/00159) to AGV, PFIS contracts (FI19/00304) to EMM, (FI21/00165) to ASA, and (FI19/00083) to CGC, and a mobility grant (MV21/00103) to EMM, from the Ministerio de Ciencia e Innovación, Spain. AJM was granted by a CSL Centenary Award.

Abstract Transcriptionally latent forms of replication-competent proviruses, present primarily in a small subset of memory CD4+ T cells, pose the primary barrier to a cure for HIV-1 infection because they are the source of the viral rebound that almost inevitably follows the interruption of antiretroviral therapy. Over the last 30 years, many of the factors essential for initiating HIV-1 transcription have been identified in studies performed using transformed cell lines, such as the Jurkat T-cell model. However, as highlighted in this review, several poorly understood mechanisms still need to be elucidated, including the molecular basis for promoter-proximal pausing of the transcribing complex and the detailed mechanism of the delivery of P-TEFb from 7SK snRNP. Furthermore, the central paradox of HIV-1 transcription remains unsolved: how are the initial rounds of transcription achieved in the absence of Tat? A critical limitation of the transformed cell models is that they do not recapitulate the transitions between active effector cells and quiescent memory T cells. Therefore, investigation of the molecular mechanisms of HIV-1 latency reversal and LRA efficacy in a proper physiological context requires the utilization of primary cell models. Recent mechanistic studies of HIV-1 transcription using latently infected cells recovered from donors and ex vivo cellular models of viral latency have demonstrated that the primary blocks to HIV-1 transcription in memory CD4+ T cells are restrictive epigenetic features at the proviral promoter, the cytoplasmic sequestration of key transcription initiation factors such as NFAT and NF-κB, and the vanishingly low expression of the cellular transcription elongation factor P-TEFb. One of the foremost schemes to eliminate the residual reservoir is to deliberately reactivate latent HIV-1 proviruses to enable clearance of persisting latently infected cells—the “Shock and Kill” strategy. For “Shock and Kill” to become efficient, effective, non-toxic latency-reversing agents (LRAs) must be discovered. Since multiple restrictions limit viral reactivation in primary cells, understanding the T-cell signaling mechanisms that are essential for stimulating P-TEFb biogenesis, initiation factor activation, and reversing the proviral epigenetic restrictions have become a prerequisite for the development of more effective LRAs.

Transcriptionally latent forms of replication-competent proviruses, present primarily in a small subset of memory CD4+ T cells, pose the primary barrier to a cure for HIV-1 infection because they are the source of the viral rebound that almost inevitably follows the interruption of antiretroviral therapy. Over the last 30 years, many of the factors essential for initiating HIV-1 transcription have been identified in studies performed using transformed cell lines, such as the Jurkat T-cell model. However, as highlighted in this review, several poorly understood mechanisms still need to be elucidated, including the molecular basis for promoter-proximal pausing of the transcribing complex and the detailed mechanism of the delivery of P-TEFb from 7SK snRNP. Furthermore, the central paradox of HIV-1 transcription remains unsolved: how are the initial rounds of transcription achieved in the absence of Tat? A critical limitation of the transformed cell models is that they do not recapitulate the transitions between active effector cells and quiescent memory T cells. Therefore, investigation of the molecular mechanisms of HIV-1 latency reversal and LRA efficacy in a proper physiological context requires the utilization of primary cell models. Recent mechanistic studies of HIV-1 transcription using latently infected cells recovered from donors and ex vivo cellular models of viral latency have demonstrated that the primary blocks to HIV-1 transcription in memory CD4+ T cells are restrictive epigenetic features at the proviral promoter, the cytoplasmic sequestration of key transcription initiation factors such as NFAT and NF-κB, and the vanishingly low expression of the cellular transcription elongation factor P-TEFb. One of the foremost schemes to eliminate the residual reservoir is to deliberately reactivate latent HIV-1 proviruses to enable clearance of persisting latently infected cells—the "Shock and Kill" strategy. For "Shock and Kill" to become efficient, effective, non-toxic latency-reversing agents (LRAs) must be discovered. Since multiple restrictions limit viral reactivation in primary cells, understanding the T-cell signaling mechanisms that are essential for stimulating P-TEFb biogenesis, initiation factor activation, and reversing the proviral epigenetic restrictions have become a prerequisite for the development of more effective LRAs. [ABSTRACT FROM AUTHOR]

Purpose of Review: HIV reservoirs are the main barrier to cure. CD4+ T cells have been extensively studied as the primary HIV-1 reservoir. However, there is substantial evidence that HIV-1-infected myeloid cells (monocytes/macrophages) also contribute to viral persistence and pathogenesis. Recent Findings: Recent studies in animal models and people with HIV-1 demonstrate that myeloid cells are cellular reservoirs of HIV-1. HIV-1 genomes and viral RNA have been reported in circulating monocytes and tissue-resident macrophages from the brain, urethra, gut, liver, and spleen. Importantly, viral outgrowth assays have quantified persistent infectious virus from monocyte-derived macrophages and tissue-resident macrophages. Summary: The myeloid cell compartment represents an important target of HIV-1 infection. While myeloid reservoirs may be more difficult to measure than CD4+ T cell reservoirs, they are long-lived, contribute to viral persistence, and, unless specifically targeted, will prevent an HIV-1 cure. [ABSTRACT FROM AUTHOR]

ABSTRACT The presence of the HIV-1 reservoir, a group of immune cells that contain intact, integrated, and replication-competent proviruses, is a major challenge to cure HIV-1. HIV-1 reservoir cells are largely unaffected by the cytopathic effects of viruses, antiviral immune responses, or antiretroviral therapy (ART). The HIV-1 reservoir is seeded early during HIV-1 infection and augmented during active viral replication. CD4+ T cells are the primary target for HIV-1 infection, and recent studies suggest that memory T follicular helper cells within the lymph node, more precisely in the B cell follicle, harbor integrated provirus, which contribute to viral rebound upon ART discontinuation. The B cell follicle, more specifically the germinal center, possesses a unique environment because of its distinct property of being partly immune privileged, potentially allowing HIV-1-infected cells within the lymph nodes to be protected from CD8+ T cells. This modified immune response in the germinal center of the follicle is potentially explained by the exclusion of CD8+ T cells and the presence of T regulatory cells at the junction of the follicle and extrafollicular region. The proviral makeup of HIV-1-infected cells is similar in lymph nodes and blood, suggesting trafficking between these compartments. Little is known about the cell-to-cell interactions, microenvironment of HIV-1-infected cells in the follicle, and trafficking between the lymph node follicle and other body compartments. Applying a spatiotemporal approach that integrates genomics, transcriptomics, and proteomics to investigate the HIV-1 reservoir and its neighboring cells in the lymph node has promising potential for informing HIV-1 cure efforts.

ABSTRACT Latency reversal and subsequent elimination of the human immunodeficiency virus-1 (HIV-1) reservoir using a combination of compounds with different mechanisms of action are considered a promising tool for HIV-1 cure. Here, we analyzed HIV-1 reservoir reduction by targeting the two host factors; inhibitor of apoptosis proteins (IAPs) and DEAD-box polypeptide 3 (DDX3) using a SMAC mimetic (SMACm) and DDX3 inhibitor (DDX3i), respectively. We observed that SMACm efficiently reactivated HIV-1 in a latency Jurkat model, which was further enhanced by DDX3 inhibition. Strikingly, this compound combination strongly decreased the proportion of latently as well as transcriptionally active infected cells in a T cell line model with a dual-reporter virus. To determine the efficacy of compounds to eradicate the HIV-1 reservoir in people living with HIV (PWH), a novel ex vivo HIV-1 reservoir reduction assay (HIVRRA) was developed. DDX3i and SMACm alone reduced the HIV-1 reservoir in peripheral blood mononuclear cells (PBMCs) from the majority of PWH, whereas notably, the SMACm/DDX3i combination reduced the HIV-1 reservoir even further with 53%–90% in all PWH analyzed, while uninfected bystander cells were not affected. Our data highlight that IAPs as well as factors involved in HIV-1 replication like DDX3 are excellent targets for HIV-1 cure strategies. We show for the first time that the combination of SMACm and DDX3i reverses viral latency and specifically eliminates the HIV-1-infected cells in vitro and ex vivo. IMPORTANCE HIV-1 continues to be a major global health challenge. Current HIV-1 treatments are effective but need lifelong adherence. An HIV-1 cure should eliminate the latent viral reservoir that persists in people living with HIV-1. Different methods have been investigated that focus on reactivation and subsequent elimination of the HIV-1 reservoir, and it is becoming clear that a combination of compounds with different mechanisms of actions might be more effective. Here, we target two host factors, inhibitor of apoptosis proteins that control apoptosis and the DEAD-box helicase DDX3, facilitating HIV mRNA transport/translation. We show that targeting of these host factors with SMAC mimetics and DDX3 inhibitors induce reversal of viral latency and eliminate HIV-1-infected cells in vitro and ex vivo.

ABSTRACTChronic inflammation and T cell dysregulation persist in individuals infected with human immunodeficiency virus type 1 (HIV-1), even after successful antiretroviral treatment. The mechanism involved is not fully understood. Here, we used Olink proteomics to comprehensively analyze the aberrant inflammation-related proteins (IRPs) in chronic HIV-1-infected individuals, including in 24 treatment-naïve individuals, 33 immunological responders, and 38 immunological non-responders. T cell dysfunction was evaluated as T cell exhaustion, activation, and differentiation using flow cytometry. We identified a cluster of IRPs (cluster 7), including CXCL11, CXCL9, TNF, CXCL10, and IL18, which was closely associated with T cell dysregulation during chronic HIV-1 infection. Interestingly, IRPs in cluster 5, including ST1A1, CASP8, SIRT2, AXIN1, STAMBP, CD40, and IL7, were negatively correlated with the HIV-1 reservoir size. We also identified a combination of CDCP1, CXCL11, CST5, SLAMF1, TRANCE, and CD5, which may be useful for distinguishing immunological responders and immunological non-responders. In conclusion, the distinct inflammatory milieu is closely associated with immune restoration of T cells, and our results provide insight into immune dysregulation during chronic HIV-1 infection.

HIV-1 replication in the gastrointestinal (GI) tract causes severe CD4+ T-cell depletion and disruption of the protective epithelial barrier in the intestinal mucosa, causing microbial translocation, the main driver of inflammation and immune activation, even in people living with HIV (PLWH) taking antiretroviral drug therapy. The higher levels of HIV DNA in the gut compared to the blood highlight the importance of the gut as a viral reservoir. CD4+ T-cell subsets in the gut differ in phenotypic characteristics and differentiation status from the ones in other tissues or in peripheral blood, and little is still known about the mechanisms by which the persistence of HIV is maintained at this anatomical site. This review aims to describe the interaction with key subsets of CD4+ T cells in the intestinal mucosa targeted by HIV-1 and the role of gut microbiome and its metabolites in HIV-associated systemic inflammation and immune activation that are crucial in the pathogenesis of HIV infection and related comorbidities. [ABSTRACT FROM AUTHOR]

HIV-1 associated colorectal cancer (HA-CRC) is one of the most understudied non-AIDS-defining cancers. In this study, we analyzed the proteome of HA-CRC and the paired remote tissues (HA-RT) through data-independent acquisition mass spectrometry (MS). The quantified proteins could differentiate the HA-CRC and HA-RT groups per PCA or cluster analyses. As a background comparison, we reanalyzed the MS data of non-HIV-1 infected CRC (non-HA-CRC) published by CPTAC. According to the GSEA results, we found that HA-CRC and non-HA-CRC shared similarly over-represented KEGG pathways. Hallmark analysis suggested that terms of antiviral response were only significantly enriched in HA-CRC. The network and molecular system analysis centered the crosstalk of IFN-associated antiviral response and cancerous pathways, which was favored by significant up-regulation of ISGylated proteins as detected in the HA-CRC tissues. We further proved that defective HIV-1 reservoir cells as represented by the 8E5 cells could activate the IFN pathway in human macrophages via horizonal transfer of cell-associated HIV-1 RNA (CA-HIV RNA) carried by extracellular vesicles (EVs). In conclusion, HIV-1 reservoir cells secreted and CA-HIV RNA-containing EVs can induce IFN pathway activation in macrophages that contributes to one of the mechanistic explanations of the systems crosstalk between antiviral response and cancerous pathways in HA-CRC.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5’ portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair.

HIV-1 latency can only be reversed in a minor fraction of human CD4+ T cells harboring genetically intact proviruses, posing a major hurdle to the elimination of the persistent reservoir. Although heterogeneous clones of latently infected cells that constitute the HIV-1 reservoir exist in a dynamic pseudo-steady state, a functional cure might be achieved by specifically targeting inducible subsets that serve as potential sources of viral rebound. Host and viral control of the transcription elongation factor P-TEFb (positive transcription elongation factor b) is a crucial rate-limiting step in the emergence of HIV-1 from latency. Therefore, comprehensively understanding its regulatory mechanisms in primary CD4+ T cells can lead to the design of more effective latency reversal agents. Sequestration of P-TEFb by 7SK snRNP (small nuclear ribonucleoprotein) is an essential feature of its regulation in primary CD4+ T cells. Both Tat and T cell signaling can remodel the ribonucleoprotein complex to facilitate the exchange of P-TEFb to proviral HIV-1. An optimal latency reversal strategy may entail HIV-1 promoter activation by either relieving epigenetic restrictions or through induction of noncanonical nuclear factor kappa B (NF-κB) signaling combined with a potent diacylglycerol mimic intended to stimulate RasGRP1-dependent biogenesis of transcriptionally active P-TEFb. Due to multiple cellular blocks in transcription, HIV-1 latency can only be reversed in a minor fraction of infected but potentially virus-producing CD4+ T cells from infected patients. Recent work shows that the biogenesis of the transcription elongation factor P-TEFb is a crucial rate-limiting step in the emergence of HIV-1 from latency. These new insights can help guide the design of more effective latency reversal agents as part of an HIV eradication strategy. Antiretroviral therapy reduces circulating HIV-1 to undetectable amounts but does not eliminate the virus due to the persistence of a stable reservoir of latently infected cells. The reservoir is maintained both by proliferation of latently infected cells and by reseeding from reactivated cells. A major challenge for the field is to find safe and effective methods to eliminate this source of rebounding HIV-1. Studies on the molecular mechanisms leading to HIV-1 latency and reactivation are being transformed using latency models in primary and patient CD4+ T cells. These studies have revealed the central role played by the biogenesis of the transcription elongation factor P-TEFb (Positive Transcription Elongation Factor b) and its recruitment to proviral HIV-1, for the maintenance of viral latency and the control of viral reactivation. [ABSTRACT FROM AUTHOR]

Supplemental Digital Content is Available in the Text. Background: Clinical trials have demonstrated noninferior viral suppression rates of selected 2-drug regimens (2DRs) over standard 3-drug regimens (3DRs). However, the effect of simplification to 2DRs on HIV-1 reservoir remains to be fully assessed. Setting: Retrospective analyses of samples of virologically suppressed people living with HIV remaining on the same 3DRs or switching to DTG + 3TC or ATV/r + 3TC 2DRs. Methods: Whole blood samples were collected at enrollment and after 48 weeks. Total HIV-1 DNA (tDNA) and intact HIV-1 DNA (iDNA) were quantified by droplet digital polymerase chain reaction and intact proviral DNA assay, respectively. Statistical analysis was performed to identify associations among variables, and multiple linear regression was used to analyze potential predictors of tDNA and iDNA changes over time. Results: Forty-seven individuals were switched to DTG + 3TC 2DR (N = 23) and ATV/r + 3TC 2DR (N = 24), while 18 remained on 3DRs. tDNA did not change either in the overall population or in the 3DR and 2DR groups. iDNA decreased significantly in the whole data set and in the overall 3DR and 2DR groups (P = 0.001, P = 0.039 and P = 0.009, respectively). iDNA, but not tDNA, was inversely correlated with the time of viral suppression (P = 0.002) and time under antiretroviral therapy (P = 0.006). Higher nadir CD4+ T-cell counts (P = 0.001) and lower zenith viral load (P = 0.02) showed an association with the decrease of iDNA, but not with tDNA. Conclusions: Both tDNA and iDNA dynamics supported noninferior efficacy of 2DRs over 3DRs. iDNA could be more informative than tDNA in analyzing the dynamics of the HIV-1 reservoir under different treatment strategies. [ABSTRACT FROM AUTHOR]

Unbiased shRNA library screens revealed that the estrogen receptor-1 (ESR-1) is a key factor regulating HIV-1 latency. In both Jurkat T cells and a Th17 primary cell model for HIV-1 latency, selective estrogen receptor modulators (SERMs, i.e., fulvestrant, raloxifene, and tamoxifen) are weak proviral activators and sensitize cells to latency-reversing agents (LRAs) including low doses of TNF-α (an NF-κB inducer), the histone deacetylase inhibitor vorinostat (soruberoylanilide hydroxamic acid, SAHA), and IL-15. To probe the physiologic relevance of these observations, leukapheresis samples from a cohort of 12 well-matched reproductive-age women and men on fully suppressive antiretroviral therapy were evaluated by an assay measuring the production of spliced envelope (env) mRNA (the EDITS assay) by next-generation sequencing. The cells were activated by T cell receptor (TCR) stimulation, IL-15, or SAHA in the presence of either β-estradiol or an SERM. β-Estradiol potently inhibited TCR activation of HIV-1 transcription, while SERMs enhanced the activity of most LRAs. Although both sexes responded to SERMs and β-estradiol, females showed much higher levels of inhibition in response to the hormone and higher reactivity in response to ESR-1 modulators than males. Importantly, the total inducible RNA reservoir, as measured by the EDITS assay, was significantly smaller in the women than in the men. We conclude that concurrent exposure to estrogen is likely to limit the efficacy of viral emergence from latency and that ESR-1 is a pharmacologically attractive target that can be exploited in the design of therapeutic strategies for latency reversal.

People living with HIV-1 and HTLV-2 concomitantly show slower CD4+ T cell depletion and AIDS progression, more frequency of the natural control of HIV-1, and lower mortality rates. A similar beneficial effect of this infection has been reported on HCV coinfection reducing transaminases, increasing the spontaneous clearance of HCV infection and delaying the development of hepatic fibrosis. Given the critical role of CD8+ T cells in controlling HIV-1 infection, we analysed the role of CD8+ T cell-mediated cytotoxic activity in coinfected individuals living with HIV-1. One hundred and twenty-eight individuals living with HIV-1 in four groups were studied: two groups with HTLV-2 infection, including individuals with HCV infection (N = 41) and with a sustained virological response (SVR) after HCV treatment (N = 25); and two groups without HTLV-2 infection, including individuals with HCV infection (N = 25) and with a sustained virological response after treatment (N = 37). We found that CD8+ T cell-mediated HIV-1 inhibition in vitro was higher in individuals with HTLV-2. This inhibition activity was associated with a higher frequency of effector memory CD8+ T cells, higher levels of granzyme A and granzyme B cytolytic enzymes, and perforin. Hence, cellular and soluble cytolytic factors may contribute to the lower HIV-1 pre-ART viral load and the HIV-1 proviral load during ART therapy associated with HTLV-2 infection. Herein, we confirmed and expanded previous findings on the role of HTLV-2 in the beneficial effect on the pathogenesis of HIV-1 in coinfected individuals. [ABSTRACT FROM AUTHOR]

Background: Among people living with HIV-1 (PHIV), immunological non-responders (INR) experience incomplete immune recovery despite suppressive antiretroviral treatment (ART), facing more severe non-AIDS events than immunological responders (IR) due to higher chronic immune activation and inflammation (cIA/I). We analyzed the HIV-1 reservoir and immunometabolism in monocytes as a source of cIA/I., Methods: Cross-sectional study in which 110 participants were enrolled: 25 treatment-naïve; 35 INR; 40 IR; and 10 healthy controls. Cell-associated HIV-1-DNA (HIV-DNA) and -RNA (HIV-RNA) were measured in FACS-isolated monocytes using digital droplet PCR. Intact, 5' deleted, and 3' deleted proviruses were quantified by the intact proviral DNA assay. Systemic inflammation, monocyte immunophenotype, and immunometabolism were characterized by immunoassays, flow cytometry, and real-time cellular bioenergetics measurements, respectively., Findings: Monocytes from INR harbor higher HIV-RNA and HIV-DNA levels than IR. HIV-RNA was found in 14/21 treatment-naïve [2512 copies/10 6 TBP (331-4666)], 17/33 INR [240 (148-589)], and 15/28 IR [144 (15-309)], correlating directly with sCD163, IP-10, GLUT1 high cells and glucose uptake, and inversely with the CD4 + /CD8 + ratio. HIV-DNA was identified in all participants with detectable HIV-RNA, with intact provirus in 9/12 treatment-naïve [13 copies/10 6 monocytes (7-44)], 8/14 INR [46 (18-67)], and 9/13 IR [9 (7-24)]. INR presented glucose metabolism alterations and mitochondrial impairment; decreased coupling efficiency and BHI, and increased mitochondrial dysfunction inversely correlating with the CD4 + /CD8 + ratio., Interpretation: HIV-RNA, more than HIV-DNA, in monocytes and their altered metabolism are factors associated with the higher cIA/I that characterize INR., Funding: This work was supported by the European Regional Development Fund, ISCIII, grant PI20/01646. Other funding sources: Instituto de Salud Carlos III through the Subprogram Miguel Servet (CP19/00159) to AGV, PFIS contracts (FI19/00304) to EMM, (FI21/00165) to ASA, and (FI19/00083) to CGC, and a mobility grant (MV21/00103) to EMM, from the Ministerio de Ciencia e Innovación, Spain. AJM was granted by a CSL Centenary Award., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Background: Reducing the number of active compounds for lifelong HIV treatment is of interest, especially to reduce potential long-term side effects. So far, available data assessing viral control, support the robustness and safety of 2DR (2-drug regimen) ART compared to 3DR. However, further in-depth investigations of the viral reservoirs are mandatory to guarantee long-term safety of these regimens regarding stable intact HIV-1 DNA copies, HIV-1 RNA transcripts and sustained immunological control., Methods: The Rumba study is the first prospective randomized controlled trial evaluating the impact of switch from 3DR to 2DR on the viral reservoir. Participants on any stable 2nd generation INSTI-based 3DR regimen with HIV-1 RNA<50 copies/ml plasma for at least 3 months were randomized to switch to dolutegravir/lamivudine (DTG/3TC, N=89) or to switch or stay on bictegravir/emtricitabine/tenofovir alafenamide (B/F/TAF, N=45). After 48 weeks, virological, immunological and metabolic parameters were evaluated., Results: We did not observe a significant difference in change over time in the mean number of intact HIV-1 DNA copies/million CD4+ T cells with DTG/3TC compared to B/F/TAF. There was no evidence in this study that switching to DTG/3TC increased the active reservoir by HIV-1 transcription. No significant changes in pro-inflammatory cytokines or major immune cell subsets were observed. Changes in exhaustion and activation of specific cellular subsets were small and bidirectional. Metabolic outcomes are similar between the treatment regimens., Conclusions: This study confirms the safety of DTG/3TC compared to B/F/TAF through viral control after in-depth investigations of the intact HIV-1 reservoir, HIV-1 transcription and inflammatory markers., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

There are approximately 38 million people globally living with Human immunodeficiency virus 1 (HIV-1) and given the tremendous success of combination antiretroviral therapy (cART) this has dramatically reduced mortality and morbidity with prevention benefits. However, HIV-1 persists during cART within the human body and re-appears upon cART interruption. This HIV-1 reservoir remains a barrier to cure with cellular sites of viral persistence not fully understood. In this study we provide evidence corroborating a recently published article in STM demonstrating the role of platelets as a novel cellular disseminator of HIV-1 particles in the setting of viral suppression. Using classical transmission electron microscopy with and without immunogold labeling, we visualize HIV-1 in both platelets and monocytes in cART suppressed HIV donors. Our study suggests that due to the close proximity of platelets and monocytes an alternative life cycle of HIV-1 cycling within monocytes and platelets without the need of active replication under cART occurs. Our findings are supported by the lack of detectable HIV-1 particles in platelets derived from HIV uninfected donors or the ‘Berlin’ patient suggesting that platelets may serve as an underappreciated hidden bearer for HIV-1 and should be considered in HIV remission studies and trials.

HIV-1 targets the monocyte/macrophage lineage and CD4+ T cells for its replication. The efficiency of infection, replication, and cell-to-cell spread differs between these cell types. These differences are caused by various factors such as viral tropism, viral proteins, host factors, and cell proliferation. However, the precise mechanisms of how macrophages influence HIV-1 infection have not been fully elucidated. Macrophages are long-lived cells susceptible to infection predominantly with R5-tropic strains of HIV-1. Although co-receptor use switches from CCR5 to CXCR4 in up to 50% of patients during AIDS progression, R5-tropic strains remain predominant in the remaining patients. Compared to HIV-1-infected T cells, infected macrophages are less susceptible to HIV-induced cytopathic effects and survive for more than a few weeks. Efforts to cure HIV-1 may be thwarted by the existence of reservoir cells that cannot be targeted by ART. Resting CD4+ T lymphocytes are thought to be the primary reservoir cells, but recent studies demonstrated that monocyte/macrophage lineage cells may also act as viral reservoirs. This review will focus on the impact of monocytes/macrophages during HIV-1 replication, the establishment of the reservoirs, and recent approaches toward HIV-1 eradication by specifically targeting monocyte/macrophage lineage cells. [ABSTRACT FROM AUTHOR]

Clinical research to achieve antiretroviral therapy-free remission requires quantitative assays of the HIV-1 reservoir. Intact proviral DNA (IPD) measurement has greater throughput than the quantitative viral outgrowth assay (QVOA). In 25 individuals with well-documented long-term viral suppression, IPD levels and infectious units per million CD4+ T cells by QVOA strongly correlated (r = 0.59, P = .002), and IPD correlated with total cell-associated HIV-1 DNA and cell-associated HIV-1 RNA (r = 0.62 and r = 0.59, P ≤ .002). IPD may provide an accessible marker of inducible replication-competent virus, total numbers of infected cells, and cellular expression of HIV-1 RNA. [ABSTRACT FROM AUTHOR]

HIV-1 reservoir size and dynamics are promising parameters to ensure the safe prescription of simplified maintenance antiretroviral therapy in chronically HIV-1 infected patients. In the SIMPL'HIV trial, HIV-1 DNA was quantified in peripheral blood mononuclear cells obtained at baseline and week 48 to investigate changes over time and evidence of a predictive relationship to maintain HIV-1 RNA <20 copies/ml. Measurements were available for 175 patients, with no differences observed between treatment strategies. Findings showed that baseline HIV-1 DNA was lower in those with durable HIV-1 RNA <20 copies/ml compared with patients with incomplete viral suppression over 48 weeks. [ABSTRACT FROM AUTHOR]

We aimed to analyze HIV-1 seroreversion caused by combination antiretroviral therapy (cART) and to explore antibody levels of anti-HIV-1 as an alternative biomarker of HIV-1 reservoir. We searched PubMed, Embase, the Cochrane Library, and Web of Science up to August 2021 for publications about the performance of HIV-1 serological assays or the association between antibody responses against HIV-1 and HIV-1 reservoirs. Potential sources of heterogeneity were explored by meta-regression analysis, including the year of publication, country, pretreatment viral load, sample size, the timing of treatment, time on cART, and principle or type of serological assay. Twenty-eight eligible studies with a total population of 1,883 were included in the meta-analysis. The pooled frequency of HIV-1 seronegativity is 38.0% (95% CI: 28.0%–49.0%) among children with vertical HIV-1 infection and cART initiation at the age of less than 6 months, while the percentage of HIV-1 seronegativity declined to 1.0% (95% CI: 0%–3.0%) when cART was initiated at the age of >6 months. For adult patients, 16.0% (95% CI: 9.0%–24.0%) of them were serologically negative when cART was initiated at acute/early infection of HIV-1, but the seronegative reaction was rarely detected when cART was started at chronic HIV-1 infection. Substantial heterogeneity was observed among the studies to estimate the frequency of HIV-1 seronegativity in the early-cART population (I 2 ≥ 70%, p < 0.05 and all), while mild heterogeneity existed for the deferred-cART subjects. Moreover, anti-HIV-1 antibody response positively correlates with HIV-1 reservoir size with a pooled rho of 0.43 (95% CI: 0.28–0.55), suggesting that anti-HIV antibody level may be a feasible biomarker of HIV-1 reservoir size. [ABSTRACT FROM AUTHOR]

Recent studies highlighted that CD8+ T cells are necessary for restraining reservoir in HIV-1-infected individuals who undergo antiretroviral therapy (ART), whereas the underlying cellular and molecular mechanisms remain largely unknown. Here, we enrolled 60 virologically suppressed HIV-1-infected individuals, to assess the correlations of the effector molecules and phenotypic subsets of CD8+ T cells with HIV-1 DNA and cell-associated unspliced RNA (CA usRNA). We found that the levels of HIV-1 DNA and usRNA correlated positively with the percentage of CCL4+CCL5- CD8+ central memory cells (TCM) while negatively with CCL4-CCL5+ CD8+ terminally differentiated effector memory cells (TEMRA). Moreover, a virtual memory CD8+ T cell (TVM) subset was enriched in CCL4-CCL5+ TEMRA cells and phenotypically distinctive from CCL4+ TCM subset, supported by single-cell RNA-Seq data. Specifically, TVM cells showed superior cytotoxicity potentially driven by T-bet and RUNX3, while CCL4+ TCM subset displayed a suppressive phenotype dominated by JUNB and CREM. In viral inhibition assays, TVM cells inhibited HIV-1 reactivation more effectively than non-TVM CD8+ T cells, which was dependent on CCL5 secretion. Our study highlights CCL5-secreting TVM cells subset as a potential determinant of HIV-1 reservoir size. This might be helpful to design CD8+ T cell-based therapeutic strategies for cure of the disease.

HIV-1 reservoir size and dynamics are promising parameters to ensure the safe prescription of simplified maintenance antiretroviral therapy in chronically HIV-1 infected patients. In the SIMPL’HIV trial, HIV-1 DNA was quantified in peripheral blood mononuclear cells obtained at baseline and week 48 to investigate changes over time and evidence of a predictive relationship to maintain HIV-1 RNA

We aimed to analyze HIV-1 seroreversion caused by combination antiretroviral therapy (cART) and to explore antibody levels of anti-HIV-1 as an alternative biomarker of HIV-1 reservoir. We searched PubMed, Embase, the Cochrane Library, and Web of Science up to August 2021 for publications about the performance of HIV-1 serological assays or the association between antibody responses against HIV-1 and HIV-1 reservoirs. Potential sources of heterogeneity were explored by meta-regression analysis, including the year of publication, country, pretreatment viral load, sample size, the timing of treatment, time on cART, and principle or type of serological assay. Twenty-eight eligible studies with a total population of 1,883 were included in the meta-analysis. The pooled frequency of HIV-1 seronegativity is 38.0% (95% CI: 28.0%–49.0%) among children with vertical HIV-1 infection and cART initiation at the age of less than 6 months, while the percentage of HIV-1 seronegativity declined to 1.0% (95% CI: 0%–3.0%) when cART was initiated at the age of >6 months. For adult patients, 16.0% (95% CI: 9.0%–24.0%) of them were serologically negative when cART was initiated at acute/early infection of HIV-1, but the seronegative reaction was rarely detected when cART was started at chronic HIV-1 infection. Substantial heterogeneity was observed among the studies to estimate the frequency of HIV-1 seronegativity in the early-cART population (I2 ≥ 70%, p < 0.05 and all), while mild heterogeneity existed for the deferred-cART subjects. Moreover, anti-HIV-1 antibody response positively correlates with HIV-1 reservoir size with a pooled rho of 0.43 (95% CI: 0.28–0.55), suggesting that anti-HIV antibody level may be a feasible biomarker of HIV-1 reservoir size.

Viral latency of human immunodeficiency virus type 1 (HIV-1) has become a major hurdle to a cure in the highly effective antiretroviral therapy (ART) era. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been demonstrated to excise or inactivate integrated HIV-1 provirus from infected cells by targeting the long terminal repeat (LTR) region. However, the guide RNAs (gRNAs) have classically avoided transcription factor binding sites (TFBSs) that are readily observed and known to be important in human promoters. Although conventionally thought unfavorable due to potential impact on human promoters, our computational pipeline identified gRNA sequences that were predicted to inactivate HIV-1 transcription by targeting the nuclear factor κB (NF-κB) binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad-spectrum activity (predicted coverage of known viral sequences). Genome-wide, unbiased identification of double strand breaks (DSBs) enabled by sequencing (GUIDE-seq) showed that the gRNAs targeting NF-κB binding sites had no detectable CRISPR-induced off-target edits in HeLa cells. 5′ LTR-driven HIV-1 transcription was significantly reduced in three HIV-1 reporter cell lines. These results demonstrate a working model to specifically target well-known TFBSs in the HIV-1 LTR that are readily observed in human promoters to reduce HIV-1 transcription with a high-level safety profile and broad-spectrum activity.

ABSTRACT Knowing the mechanisms that govern the persistence of infected CD4+ subpopulations could help us to design new therapies to cure HIV-1 infection. We evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ subpopulations from 14 HIV-1-infected individuals on antiretroviral therapy to analyze its relationship with HIV-1 transcription, immune activation, and cell proliferation. A unique large blood donation was used to isolate CD4, CD4 resting (CD4r), CD4 activated (CD4a), T naive (TN), T stem cell memory (TSCM), T central memory (TCM), T transitional memory (TTM), T effector memory (TEM), circulating T follicular helper (cTFH), TCD20, TCD32, and resting memory TCD2high (rmTCD2high) cells. HIV-1 DNA measured by droplet digital PCR ranged from 3,636 copies/106 in TTM to 244 in peripheral blood mononuclear cells (PBMCs), with no subpopulation standing out for provirus enrichment. Importantly, all the subpopulations harbored intact provirus by intact provirus DNA assay (IPDA). TCD32, cTFH, and TTM had the highest levels of HIV-1 transcription measured by fluorescent in situ hybridization with flow cytometry (FISH/flow), but without reaching statistical differences. The subpopulations more enriched in provirus had a memory phenotype, were less activated (measured by CD38+/HLA-DR+), and expressed more programmed cell death 1 (PD-1). Conversely, subpopulations transcribing more HIV-1 RNA were not necessarily enriched in provirus and were more activated (measured by CD38+/HLA-DR+) and more proliferative (measured by Ki-67). In conclusion, the HIV reservoir is composed of a mosaic of subpopulations contributing to the HIV-1 persistence through different mechanisms such as susceptibility to infection, provirus intactness, or transcriptional status. The narrow range of reservoir differences between the different blood cell subsets tested suggests limited efficacy in targeting only specific cell subpopulations during HIV-1 cure strategies. IMPORTANCE The main barrier for HIV-1 cure is the presence of latently infected CD4+ T cells. Although various cell subpopulations have been identified as major HIV-1 reservoir cells, the relative contribution of infected CD4 subpopulations in the HIV-1 reservoir remains largely unknown. Here, we evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ T-cell subpopulations in peripheral blood from HIV-1-infected individuals under suppressive antiretroviral therapy. We found that the HIV-1 reservoir is composed of a mosaic of cell subpopulations, with heterogeneous proviral DNA, HIV-1 transcription, and activation status. Hence, each cell subpopulation contributes to the HIV-1 persistence through different mechanisms such as susceptibility to infection, rates of intact provirus, transcriptional status or half-life. This research provides new insights into the composition of the HIV-1 reservoir, suggesting that, to be effective, eradication strategies must simultaneously target multiple cell subpopulations.

The presence of the HIV-1 reservoir, a group of immune cells that contain intact, integrated, and replication-competent proviruses, is a major challenge to cure HIV-1. HIV-1 reservoir cells are largely unaffected by the cytopathic effects of viruses, antiviral immune responses, or antiretroviral therapy (ART). The HIV-1 reservoir is seeded early during HIV-1 infection and augmented during active viral replication. CD4+ T cells are the primary target for HIV-1 infection, and recent studies suggest that memory T follicular helper cells within the lymph node, more precisely in the B cell follicle, harbor integrated provirus, which contribute to viral rebound upon ART discontinuation. The B cell follicle, more specifically the germinal center, possesses a unique environment because of its distinct property of being partly immune privileged, potentially allowing HIV-1-infected cells within the lymph nodes to be protected from CD8+ T cells. This modified immune response in the germinal center of the follicle is potentially explained by the exclusion of CD8+ T cells and the presence of T regulatory cells at the junction of the follicle and extrafollicular region. The proviral makeup of HIV-1-infected cells is similar in lymph nodes and blood, suggesting trafficking between these compartments. Little is known about the cell-to-cell interactions, microenvironment of HIV-1-infected cells in the follicle, and trafficking between the lymph node follicle and other body compartments. Applying a spatiotemporal approach that integrates genomics, transcriptomics, and proteomics to investigate the HIV-1 reservoir and its neighboring cells in the lymph node has promising potential for informing HIV-1 cure efforts., Competing Interests: The authors declare no conflict of interest.

There are approximately 38 million people globally living with Human immunodeficiency virus 1 (HIV-1) and given the tremendous success of combination antiretroviral therapy (cART) this has dramatically reduced mortality and morbidity with prevention benefits. However, HIV-1 persists during cART within the human body and re-appears upon cART interruption. This HIV-1 reservoir remains a barrier to cure with cellular sites of viral persistence not fully understood. In this study we provide evidence corroborating a recently published article in STM demonstrating the role of platelets as a novel cellular disseminator of HIV-1 particles in the setting of viral suppression. Using classical transmission electron microscopy with and without immunogold labeling, we visualize HIV-1 in both platelets and monocytes in cART suppressed HIV donors. Our study suggests that due to the close proximity of platelets and monocytes an alternative life cycle of HIV-1 cycling within monocytes and platelets without the need of active replication under cART occurs. Our findings are supported by the lack of detectable HIV-1 particles in platelets derived from HIV uninfected donors or the 'Berlin' patient suggesting that platelets may serve as an underappreciated hidden bearer for HIV-1 and should be considered in HIV remission studies and trials. [ABSTRACT FROM AUTHOR]

BackgroundThe stability of the human immunodeficiency virus type 1 (HIV-1) reservoir and the contribution of cellular proliferation to the maintenance of the reservoir during treatment are uncertain. Therefore, we conducted a longitudinal analysis of HIV-1 in T-cell subsets in different tissue compartments from subjects receiving effective antiretroviral therapy (ART).MethodsUsing single-proviral sequencing, we isolated intracellular HIV-1 genomes derived from defined subsets of CD4(+) T cells from peripheral blood, gut-associated lymphoid tissue and lymph node tissue specimens from 8 subjects with virologic suppression during long-term ART at 2 time points (time points 1 and 2) separated by 7-9 months.ResultsDNA integrant frequencies were stable over time (

People living with HIV-1 and HTLV-2 concomitantly show slower CD4+ T cell depletion and AIDS progression, more frequency of the natural control of HIV-1, and lower mortality rates. A similar beneficial effect of this infection has been reported on HCV coinfection reducing transaminases, increasing the spontaneous clearance of HCV infection and delaying the development of hepatic fibrosis. Given the critical role of CD8+ T cells in controlling HIV-1 infection, we analysed the role of CD8+ T cell-mediated cytotoxic activity in coinfected individuals living with HIV-1. One hundred and twenty-eight individuals living with HIV-1 in four groups were studied: two groups with HTLV-2 infection, including individuals with HCV infection (N = 41) and with a sustained virological response (SVR) after HCV treatment (N = 25); and two groups without HTLV-2 infection, including individuals with HCV infection (N = 25) and with a sustained virological response after treatment (N = 37). We found that CD8+ T cell-mediated HIV-1 inhibition in vitro was higher in individuals with HTLV-2. This inhibition activity was associated with a higher frequency of effector memory CD8+ T cells, higher levels of granzyme A and granzyme B cytolytic enzymes, and perforin. Hence, cellular and soluble cytolytic factors may contribute to the lower HIV-1 pre-ART viral load and the HIV-1 proviral load during ART therapy associated with HTLV-2 infection. Herein, we confirmed and expanded previous findings on the role of HTLV-2 in the beneficial effect on the pathogenesis of HIV-1 in coinfected individuals.

The establishment of HIV-1 latency has hindered an HIV-1 cure. "Shock and kill" strategies to target this reservoir aim to induce the latent provirus with latency- reversing agents (LRAs). However, recent studies have shown that the majority of the intact HIV-1 viral reservoir found in antiretroviral therapy (ART)-suppressed HIV-infected individuals is not inducible. We sought to understand whether this noninducible reservoir is established, and thus able to be studied, in an in vitro primary TCM cell model of latency. Furthermore, we wanted to expand this model system to include R5-tropic and non-B-subtype viruses. To that end, we generated our TCM cell model of latency with an R5 subtype B virus, AD8, and an R5 subtype C virus, MJ4. Our results demonstrate that both intact and defective proviruses are generated in this model. Less than 50% of intact proviruses are inducible regardless of viral strain in the context of maximal stimulation through the T cell receptor (TCR) or with different clinically relevant LRAs, including the histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) and MS-275, the protein kinase C (PKC) agonist ingenol 3,20-dibenzoate, or the second mitochondria-derived activator of caspase (SMAC) mimetic AZD-5582. Our findings suggest that current LRA strategies are insufficient to effectively reactivate intact latent HIV-1 proviruses in primary CD4 TCM cells and that the mechanisms involved in the generation of the noninducible HIV-1 reservoir can be studied using this primary in vitro model. [ABSTRACT FROM AUTHOR]

Despite potent combination antiretroviral therapy, HIV-1 infection persists due to irreversible integration of the virus in long-living cells of the immune system. The main focus of HIV-1 cure strategies has been on HIV-1 eradication, yet without great success so far. Therefore, HIV-1 remission or a functional cure, whereby the virus is silenced rather than eradicated, is considered as an alternative strategy. Elite controllers, individuals who spontaneously control HIV-1, may point us the way toward a functional HIV-1 cure. In order to achieve such a cure, a profound understanding of the mechanisms controlling HIV-1 expression and silencing is needed. In recent years, evidence has grown that the site of integration as well as the chromatin landscape surrounding the integration site affects the transcriptional state of the provirus. Still, at present, the impact of integration site selection on the establishment and maintenance of the HIV-1 reservoirs remains poorly understood. The discovery of LEDGF/p75 as a binding partner of HIV-1 integrase has led to a better understanding of integration site selection. LEDGF/p75 is one of the important determinants of integration site selection and targets integration toward active genes. In this review, we will provide an overview of the most important determinants of integration site selection. Secondly, we will discuss the chromatin landscape at the integration site and its implications on HIV-1 gene expression and silencing. Finally, we will discuss how interventions that affect integration site selection or modifications of the chromatin could yield a functional cure of HIV-1 infection. [ABSTRACT FROM AUTHOR]

Low-level viremia (LLV) ranging from 50 to 1,000 copies/ml is common in most HIV-1-infected patients receiving antiretroviral therapy (ART). However, the source of LLV and the impact of LLV on the HIV-1 reservoir during ART remain uncertain. We hypothesized that LLV may arise from the HIV reservoir and its occurrence affect the composition of the reservoir after LLV episodes. Accordingly, we investigated the genetic linkage of sequences obtained from plasma at LLV and pre-ART time points and from peripheral blood mononuclear cells (PBMCs) at pre-ART, pre-LLV, LLV, and post-LLV time points. We found that LLV sequences were populated with a predominant viral quasispecies that accounted for 67.29%∼100% of all sequences. Two episodes of LLV in subject 1, spaced 6 months apart, appeared to have originated from the stochastic reactivation of latently HIV-1-infected cells. Moreover, 3.77% of pre-ART plasma sequences were identical to 67.29% of LLV-3 plasma sequences in subject 1, suggesting that LLV may have arisen from a subset of cells that were infected before ART was initiated. No direct evidence of sequence linkage was found between LLV viruses and circulating cellular reservoirs in all subjects. The reservoir size, diversity, and divergence of the PBMC DNA did not differ significantly between the pre- and post-LLV sampling points ( P > 0.05), but the composition of viral reservoir quasispecies shifted markedly before and after LLV episodes. Indeed, subjects with LLV had a higher total PBMC DNA level, greater viral diversity, a lower proportion of variants with identical sequences detected at two or more time points, and a shorter variant duration during ART compared with subjects without LLV. Overall, our findings suggested that LLV viruses may stem from an unidentified source other than circulating cellular reservoirs. LLV episodes may introduce great complexity into the HIV reservoir, which brings challenges to the development of treatment strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sun, Zhang, Kong, Li, Zhang, An, Ding, Shang and Han.)

Although HIV-1 has evolved from a deadly to a chronic disease over the past 20 years, an HIV-1 cure is still lacking due to the presence of persisting cellular viral reservoirs which are spread throughout the body in different anatomical compartments. Hence, the identification and characterization of these HIV-1 reservoirs were the focus of many studies during the past decades. In this review, a systematic literature screening and text mining approach were implemented to assess the evolution in experimental design of these HIV-1 reservoir studies. For this purpose, the online databases PubMed, Web of Science, and ClinicalTrials.gov were consulted and 1768 articles were identified, of which 106 are included in this review. We observed several evolutions that indicate a more structured approach of recent HIV-1 reservoir studies. This includes the use of well-characterized patient cohorts, tissue sampling at several time points and anatomical compartments, the inclusion of patients with different treatment status (on and off antiretroviral therapy), and the implementation of state-of-the-art research techniques such as single genome sequencing. In addition, there is an increased interest and sampling of lymphoid tissues and cerebrospinal fluid together with methods to investigate cellular subsets and HIV-1 sequences. Overall, this review describes an observed shift from detecting and quantifying HIV-1 toward a qualitative in-depth assessment of anatomical reservoirs and cellular subsets playing a role in HIV-1 persistence/latency. These trends coincide with the evolution in focus from controlling HIV-1 replication by currently available antiretroviral therapy toward HIV-1 curative strategies. [ABSTRACT FROM AUTHOR]

Background: Human IL-32 is a polyfunctional cytokine that was initially reported to inhibit HIV-1 infection. However, recent data suggest that IL-32 may enhance HIV-1 replication by activating the HIV-1 primary targets, CD4+ T-cells. Indeed, IL-32 is expressed in multiple isoforms, some of which are proinflammatory, whereas others are anti-inflammatory. Setting and Methods: Here, we aimed to determine the relative expression of IL-32 isoforms and to test their inflammatory nature and potential to induce HIV-1 production in latently infected cells from virologically suppressed HIV-infected individuals. IL-32 and other cytokines were quantified from plasma and supernatant of CD4+ T-cells by ELISA. Transcripts of IL-32 isoforms were quantified by qRT-PCR in peripheral blood mononuclear cells. The impact of recombinant human IL-32 isoforms on HIV-1 transcription was assessed in CD4+ T-cells from HIV-1+cART+ individuals by qRT-PCR. Results: All IL-32 isoforms were significantly upregulated in HIV- 1+cART+ compared to HIVneg individuals with IL-32β representing the dominantly expressed isoform, mainly in T-cells and NK-cells. At the functional level, although IL-32γ induced typical proinflammatory cytokines (IL-6 and IFN-γ) in TCR-activated CD4+ T-cells, IL-32α showed an anti-inflammatory profile by inducing IL-10 but not IL-6 or IFN-γ. However, IL-32β showed a dual phenotype by inducing both pro- and anti-inflammatory cytokines. Interestingly, consistent with its highly pro-inflammatory nature, IL-32γ, but not IL-32α or IL-32β, induced HIV-1 production in latently infected CD4U T-cells isolated from combined antiretroviral therapy--treated individuals. Conclusions: Our data report on the differential expression of IL-32 isoforms and highlight the potential role of IL-32, particularly the γ isoform, in fueling persistent inflammation and transcription of viral reservoir in HIV-1 infection. [ABSTRACT FROM AUTHOR]

• Detection of cell-associated HIV-1 parameters to monitor HIV-1 persistence. • ddPCR-based assays validated to meet CLIA/GCLP requirements. • Detection across multiple HIV-1 subtypes. • Sensitive assays for cell-associated HIV-1 DNA, unspliced RNA and 2-LTR circle. Cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA (usRNA) are important virological parameters for monitoring HIV-1 persistence and activation of latent HIV-1. Assays fully validated by CLIA and/or GCLP standards are needed for future clinical trials that seek to evaluate treatments directed towards HIV-1 cure. To determine performance characteristics of sensitive, moderate-throughput, digital droplet PCR (ddPCR) assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA that can detect a broad range of HIV-1 M-group subtypes. To evaluate linearity, limit of detection, precision, and accuracy of each assay, contrived specimens were analyzed in a background of uninfected PBMC. Detection breadth was evaluated by in silico analysis of primer and probes sets and analysis of material harvested from PBMC infected in vitro with various HIV-1 subtypes. A cohort of clinical specimens from viremic and virologically suppressed individuals was analyzed to demonstrate applicability to clinical research. The empirically determined limit of detection of these assays was 29, 7, and 60 copies per million PBMC for HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA, respectively. The assays detect a broad range of HIV-1 M-group subtypes. Finally, analysis of clinical specimens demonstrate that these assays can detect low levels of cell-associated HIV-1 DNA, HIV-1 usRNA, and HIV-1 2-LTR circle and correlate with clinical histories and viral loads of untreated and antiretroviral treated individuals. We report the clinical validation of three HIV reservoir assays with broad HIV-1 coverage for future cure studies. [ABSTRACT FROM AUTHOR]

Antiretroviral therapy (ART) suppresses HIV-1 replication but does not eradicate the virus. Persistence of HIV-1 latent reservoirs in ART-treated individuals is considered the main obstacle to achieving an HIV-1 cure. However, these HIV-1 reservoirs are not transcriptionally silent, and viral transcripts can be detected in most ART-treated individuals. HIV-1 latency is regulated at the transcriptional and at multiple post-transcriptional levels. Here, we review recent insights into the possible contribution of viral RNA processing to the persistence of HIV-1 reservoirs, and discuss the clinical implications of persistence of viral RNA species in ART-treated individuals.

Background: If analytical antiretroviral-treatment (ART) interruption (ATI) might significantly impact quantitative or qualitative peripheral-total HIV-DNA is still debated. Methods: Six chronically HIV-1 infected patients enrolled in APACHE-study were analysed for peripheral-total HIV-DNA and residual viremia, major-resistance-mutations (MRMs) and C2-V3-C3 evolution at pre-ATI (T1), during ATI (T2) and at achievement of virological success after ART-resumption (post-ATI, T3). These data were obtained at three comparable time-points in five chronically HIV-1 infected patients on suppressive ART for ≥1 year, enrolled in MODAt-study. Results: At T1, APACHE and MODAt individuals had similar peripheral-total HIV-DNA and residual viremia (p = 0.792 and 0.662, respectively), and no significant changes for these parameters were observed between T1 and T3 in both groups. At T1, 4/6 APACHE and 2/5 MODAt carried HIV-DNA MRMs. MRMs disappeared at T3 in 3/4 APACHE. All disappearing MRMs were characterized by T1 intra-patient prevalence p = 0.04), while no significant changes were found in MODAt. Accordingly, maximum likelihood trees (bootstrap > 70%) and genealogical sorting indices (GSI > 0.50 with p-value < 0.05) showed that T1 C2-V3-C3 DNA sequences were distinct from T2 and T3 viruses in 4/6 APACHE. Virus populations at all three time-points were highly interspersed in MODAt. Conclusions: This pilot study indicates that short ATI does not alter peripheral-total HIV-DNA burden and residual viremia, but in some cases could cause a genetic diversification of peripheral viral reservoir in term of both MRMs rearrangement and viral evolution.

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

Determining the HIV-1 reservoir size in infected individuals is of great importance for improvement of their treatment. Plasma trans-activation response element (TAR) RNA has been suggested as one of the possible biomarkers. TAR RNA is produced during non-processive transcription in HIV-1 productively infected and latent T cells. Here, plasma samples and paired exosome samples of 55 subjects from the observational SCOPE cohort were analysed for the presence of TAR RNA. First, a PCR-based assay was optimized, which provided 100% specificity and 100% sensitivity in differentiating HIV-1 infected non-controllers from uninfected individuals. Next, TAR RNA was detected in the plasma of 63% of aviremic HIV-1–infected patients, who were either treated with antiretroviral therapy or were elite controllers. Although TAR RNA levels did not correlate with patient gender, age, CD4 levels, CD8 levels, they tended to correlate with CD4/CD8 ratio (P = 0.047). This study is the first to investigate plasma TAR RNA in a relatively large cohort of HIV-1–infected patients. We additionally show that the TAR RNA molecules in the plasma of aviremic patients are not limited to exosomes.

The presence of latent HIV-1 in infected individuals represents a major barrier preventing viral eradication. For that reason, reactivation of latent viruses in the presence of antiretroviral regimens has been proposed as a therapeutic strategy to achieve remission. We screened for small molecules and identified several benzotriazole derivatives with the ability to reactivate latent HIV-1. In the presence of IL-2, benzotriazoles reactivated and reduced the latent reservoir in primary cells, and, remarkably, viral reactivation was achieved without inducing cell proliferation, T cell activation, or cytokine release. Mechanistic studies showed that benzotriazoles block SUMOylation of phosphorylated STAT5, increasing STAT5’s activity and occupancy of the HIV-1 LTR. Our results identify benzotriazoles as latency reversing agents and STAT5 signaling and SUMOylation as targets for HIV-1 eradication strategies. These compounds represent a different direction in the search for “shock and kill” therapies.

The so-called shock and kill therapies aim to combine HIV-1 reactivation by latency-reversing agents (LRA) with immune clearance to purge the HIV-1 reservoir. The clinical use of LRA has demonstrated detectable perturbations in the HIV-1 reservoir without measurable reductions to date. Consequently, fundamental questions concerning the limitations of the recognition and killing of LRA-reactivated cells by effector cells such as CD8+ T cells remain to be answered. Here, we developed a novel experimental framework where we combine the use of cytotoxic CD8+ T-cell lines and ex vivo CD8+ T cells from HIV-1-infected individuals with functional assays of LRA-inducible reactivation to delineate immune barriers to clear the reservoir. Our results demonstrate the potential for early recognition and killing of reactivated cells by CD8+ T cells. However, the potency of LRAs when crossing the barrier for antigen presentation in target cells, together with the lack of expression of inhibitory receptors in CD8+ T cells, are critical events to maximize the speed of recognition and the magnitude of the killing of LRA-inducible provirus. Taken together, our findings highlight direct limitations in LRA potency and CD8+ T cell functional status to succeed in the cure of HIV-1 infection.

Importance: HIV-1 continues to be a major global health challenge. Current HIV-1 treatments are effective but need lifelong adherence. An HIV-1 cure should eliminate the latent viral reservoir that persists in people living with HIV-1. Different methods have been investigated that focus on reactivation and subsequent elimination of the HIV-1 reservoir, and it is becoming clear that a combination of compounds with different mechanisms of actions might be more effective. Here, we target two host factors, inhibitor of apoptosis proteins that control apoptosis and the DEAD-box helicase DDX3, facilitating HIV mRNA transport/translation. We show that targeting of these host factors with SMAC mimetics and DDX3 inhibitors induce reversal of viral latency and eliminate HIV-1-infected cells in vitro and ex vivo ., Competing Interests: M.A., J.B., C.Z., and A.T. are employees of First Health Pharmaceuticals B.V.