Latent HIV Reservoir Research Articles

A Widely-Distributed Hiv-1 Provirus Elimination Assay to Evaluate Latency-Reversing Agents in Vitro

Persistence of HIV-1 latent reservoir cells during antiretroviral therapy is a major obstacle for curing HIV-1. Latency-reversing agents (LRAs) are under development to reactivate and eradicate latently infected cells; however, there are few useful models for evaluating LRA activity in vitro . Here we established a long-term cell culture system harboring thousands of different HIV-1-infected cell clones with a wide distribution of HIV-1 provirus similar to that observed in vivo . A combination of an LRA and antiretroviral therapy (ART) significantly reduced viral rebound upon treatment interruption. Experimental results and mathematical modeling demonstrated that addition of LRA to ART showed latency-reversing effect and contributed to the eradication of replication competent HIV-1. The widely distributed intact provirus elimination (WIPE) assay can be used to optimize therapeutic against HIV-1 latency and investigate mechanistic insights into the clonal selection of heterogeneous HIV-1-infected cells.

The multifaceted nature of HIV latency.

Although antiretroviral therapies (ARTs) potently inhibit HIV replication, they do not eradicate the virus. HIV persists in cellular and anatomical reservoirs that show minimal decay during ART. A large number of studies conducted during the past 20 years have shown that HIV persists in a small pool of cells harboring integrated and replication-competent viral genomes. The majority of these cells do not produce viral particles and constitute what is referred to as the latent reservoir of HIV infection. Therefore, although HIV is not considered as a typical latent virus, it can establish a state of nonproductive infection under rare circumstances, particularly in memory CD4+ T cells, which represent the main barrier to HIV eradication. While it was originally thought that the pool of latently infected cells was largely composed of cells harboring transcriptionally silent genomes, recent evidence indicates that several blocks contribute to the nonproductive state of these cells. Here, we describe the virological and immunological factors that play a role in the establishment and persistence of the pool of latently infected cells and review the current approaches aimed at eliminating the latent HIV reservoir.

A delay model for persistent viral infections in replicating cells.

Persistently infecting viruses remain within infected cells for a prolonged period of time without killing the cells and can reproduce via budding virus particles or passing on to daughter cells after division. The ability for populations of infected cells to be long-lived and replicate viral progeny through cell division may be critical for virus survival in examples such as HIV latent reservoirs, tumor oncolytic virotherapy, and non-virulent phages in microbial hosts. We consider a model for persistent viral infection within a replicating cell population with time delay in the eclipse stage prior to infected cell replicative form. We obtain reproduction numbers that provide criteria for the existence and stability of the equilibria of the system and provide bifurcation diagrams illustrating transcritical (backward and forward), saddle-node, and Hopf bifurcations, and provide evidence of homoclinic bifurcations and a Bogdanov-Takens bifurcation. We investigate the possibility of long term survival of the infection (represented by chronically infected cells and free virus) in the cell population by using the mathematical concept of robust uniform persistence. Using numerical continuation software with parameter values estimated from phage-microbe systems, we obtain two parameter bifurcation diagrams that divide parameter space into regions with different dynamical outcomes. We thus investigate how varying different parameters, including how the time spent in the eclipse phase, can influence whether or not the virus survives.

Evaluation of HIV-1 latency reversal and antibody-dependent viral clearance by quantification of singly spliced HIV-1 vpu/env mRNA.

The latent reservoir of HIV-1 is a major barrier for viral eradication. Potent HIV-1 broadly neutralizing antibodies (bNabs) have been used to prevent and treat HIV-1 infections in animal models and clinical trials. Combination of bNabs and latency-reversing agents (LRAs) is considered a promising approach for HIV-1 eradication. PCR-based assays that can rapidly and specifically measure singly spliced HIV-1 vpu/env mRNA are needed to evaluate the induction of the viral envelope production at the transcription level and bNab-mediated reservoir clearance. Here we reported a PCR-based method to accurately quantify the production of intracellular HIV-1 vpu/env mRNA. With the vpu/env assay, we determined the LRA combinations that could effectively induce vpu/env mRNA production in CD4+ T cells from ART-treated individuals. None of the tested LRAs were effective alone. A comparison between the quantitative viral outgrowth assay (Q-VOA) and the vpu/env assay showed that vpu/env mRNA production was closely associated with the reactivation of replication-competent HIV-1, suggesting that vpu/env mRNA was mainly produced by intact viruses. Finally, antibody-mediated in vitro killing in HIV-1-infected humanized mice demonstrated that the vpu/env assay could be used to measure the reduction of infected cells in tissues and was more accurate than the commonly used gag-based PCR assay which measured unspliced viral genomic RNA. In conclusion, the vpu/env assay allows convenient and accurate assessment of HIV-1 latency reversal and bNab-mediated therapeutic strategies.ImportanceHIV-1 persists in individuals on antiretroviral therapy (ART) due to the long-lived cellular reservoirs that contain dormant viruses. Recent discoveries of HIV-1-specific broadly neutralizing antibodies (bNabs) targeting HIV-1 Env protein rekindled the interest in antibody-mediated elimination of latent HIV-1. Latency-reversing agents (LRAs) together with HIV-1 bNabs is a possible strategy to clear residual viral reservoirs, which makes the evaluation of HIV-1 Env expression upon LRA treatment critical. We developed a PCR-based assay to quantify the production of intracellular HIV-1 vpu/env mRNA. Using patient CD4+ T cells, we found that induction of HIV-1 vpu/env mRNA required a combination of different LRAs. Using in vitro, ex vivo and humanized mouse models, we showed that the vpu/env assay could be used to measure antibody efficacy in clearing HIV-1 infection. These results suggest that the vpu/env assay can accurately evaluate HIV-1 reactivation and bNab-based therapeutic interventions.

Are Fc Gamma Receptor Polymorphisms Important in HIV-1 Infection Outcomes and Latent Reservoir Size?

Fc gamma receptors (FcγR) are cell surface glycoproteins which trigger specific effector-cell responses when cross-linked with the Fc portions of immunoglobulin (IgG) antibodies. During HIV-1 infection, the course of disease progression, ART response, and viral reservoir size vary in different individuals. Several factors may account for these differences; however, Fc gamma receptor gene polymorphisms, which influence receptor binding to IgG antibodies, are likely to play a key role. FcγRIIa (CD32) was recently reported as a potential marker for latent HIV reservoir, however, this assertion is still inconclusive. Whether FcγR polymorphisms influence the size of the viral reservoir, remains an important question in HIV cure studies. In addition, potential cure or viral suppression methods such as broadly neutralizing antibody (bNAbs) may depend on FcγRs to control the virus. Here, we discuss the current evidence on the potential role played by FcγR polymorphisms in HIV-1 infection, treatment and vaccine trial outcomes. Importantly, we highlight contrasting findings that may be due to multiple factors and the relatively limited data from African populations. We recommend further studies especially in sub-Saharan Africa to confirm the role of FcγRIIa in the establishment of latent reservoir and to determine their influence in therapies involving bNAbs.

Anti-HIV-1 ADCC and HIV-1 Env Can Be Partners in Reducing Latent HIV Reservoir.

BackgroundPersistence of HIV reservoir even in suppressive ART is the key obstacle in HIV-1 cure. We evaluated the ability of HIV-1 C Env to reactivate the latently infected resting memory CD4 cells and the ability of polyclonal HIV antibodies mediating ADCC to lyse the reactivated targets.MethodologyHIV-1 antibodies from 25 HIV infected individuals (14 ADCC responders and 11 non-responders) were tested against the Env-C reactivated primary cells; CD4+ and CD4+CD45RO+ memory T cells in the presence of autologous or heterologous effector cells using multicolor flow cytometry. The frequencies of p24+ve target cells were measured to determine the reactivation and antibody mediated lysis.ResultsIncrease in the frequency of p24 expressing cells (P < 0.01 in all cases) after Env-C stimulation of target cells indicated reactivation. When these reactivated targets were mixed with effector cells and HIV-1 antibodies, the frequencies of p24 expressing targets were decreased significantly when the ADCC mediating antibodies (P < 0.01 in all cases) were added but not when the antibodies from ADCC non-responders or HIV negative individuals were added. In parallel, the NK cell activation was also increased only when ADCC mediating antibodies were added.ConclusionThe study showed that the HIV-1 Env could act as latency reversal agent (LRA), and only ADCC mediating antibodies could lyse the reactivated HIV reservoirs. The short stimulation cycle used in this study could be useful in testing LRAs as well as immune mediated lysis of reactivated reservoirs. The observations have further implication in designing antibody mediated immunotherapy for eradication of latent HIV reservoir.

Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir

An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). We used single-cell FISH-Flow technology to characterise the contribution of viral RNA to inducing cell death. The pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3 and upregulation of IFNβ. DDX3 inhibition also resulted in the downregulation of BIRC5, critical to cell survival during HIV-1 infection, and selectively induced apoptosis in viral RNA-expressing CD4+ T cells but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir by quantitation of HIV-1 RNA, by FISH-Flow, RT-qPCR and TILDA. This study provides proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards the elimination of the inducible reservoir.

CpG Methylation Profiles of HIV-1 Pro-Viral DNA in Individuals on ART.

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5′ LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5′ LTR promoter.

T cell immune discriminants of HIV reservoir size in a pediatric cohort of perinatally infected individuals.

The size of the latent HIV reservoir is associated with the timing of therapeutic interventions and overall health of the immune system. Here, we demonstrate that T cell phenotypic signatures associate with viral reservoir size in a cohort of HIV vertically infected children and young adults under durable viral control, and who initiated anti-retroviral therapy (ART) <2 years old. Flow cytometry was used to measure expression of immune activation (IA), immune checkpoint (ICP) markers, and intracellular cytokine production after stimulation with GAG peptides in CD4 and CD8 T cells from cross-sectional peripheral blood samples. We also evaluated the expression of 96 genes in sort-purified total CD4 and CD8 T cells along with HIV-specific CD4 and CD8 T cells using a multiplexed RT-PCR approach. As a measure of HIV reservoir, total HIV-DNA quantification by real-time PCR was performed. Poisson regression modeling for predicting reservoir size using phenotypic markers revealed a signature that featured frequencies of PD-1+CD4 T cells, TIGIT+CD4 T cells and HIV-specific (CD40L+) CD4 T cells as important predictors and it also shows that time of ART initiation strongly affects their association with HIV-DNA. Further, gene expression analysis showed that the frequencies of PD-1+CD4 T cells associated with a CD4 T cell molecular profile skewed toward an exhausted Th1 profile. Our data provide a link between immune checkpoint molecules and HIV persistence in a pediatric cohort as has been demonstrated in adults. Frequencies of PD-1+ and TIGIT+CD4 T cells along with the frequency of HIV-specific CD4 T cells could be associated with the mechanism of viral persistence and may provide insight into potential targets for therapeutic intervention.

Measuring the size and decay dynamics of the HIV-1 latent reservoir

Measuring HIV-1 latent reservoir is essential for HIV-1 cure strategies. Levy et al.1 developed a multiplex droplet digital PCR (ddPCR) assay—5-target intact proviral DNA assay—to detect multiple regions of HIV-1 proviral genome and increase accuracy.

Initiation of Antiretroviral Therapy during Primary HIV Infection: Effects on the Latent HIV Reservoir, Including on Analytic Treatment Interruptions.

Antiretroviral therapy (ART) inhibits HIV replication but does not eradicate the latent reservoir. The previous research suggests that earlier ART initiation provides benefit on limiting reservoir size, but timing and extent of this effect remain unclear. Analytic treatment interruption (ATI) may be used to demonstrate HIV remission, but whether early ART also improves likelihood or duration of even temporary virologic remission is unclear. This review seeks to answer both questions. We performed a systematic review and analysis following Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines and included 21 interventional or observational studies with sufficient HIV reservoir outcomes. We also aggregated reservoir outcomes and transformed data into approximate measurements of total HIV DNA per million peripheral blood mononuclear cells and analyzed the correlation between timing of ART initiation and reservoir size. People living with HIV who initiate ART in primary infection maintain smaller reservoirs on suppressive ART than those who initiate treatment during chronic infection. The reduction of reservoir is most pronounced when ART is started within 2 weeks of HIV acquisition. Across studies, we found a moderately strong association between longer time to ART initiation and reservoir size, which was strongest when measured after 1 year on ART (Pearson's r = 0.69, p = 0.0003). After ATI, larger pre-ATI reservoir size predicts shorter time to viral rebound. Early ART may also facilitate long-term control of viremia. Although achieving sustained HIV remission will require further interventions, initiating ART very early in infection could limit the extent of the reservoir and also lead to post-ATI control in rare cases.

The Intact Non-Inducible Latent HIV-1 Reservoir is Established In an In Vitro Primary TCM Cell Model of Latency.

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 ART-suppressed HIV infected individuals is not inducible. We sought to understand whether this non-inducible reservoir is established, and thus able to be studied, in an in vitro primary TCM 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 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 TCR or with different clinically relevant LRAs including the HDAC inhibitors SAHA and MS-275, the PKC agonist Ingenol 3,20-dibenzoate or the 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 non-inducible HIV-1 reservoir can be studied using this primary in vitro model.Importance: HIV-1 establishes a latent reservoir that persists under antiretroviral therapy. Antiretroviral therapy is able to stop the spread of the virus and the progression of the disease but does not target this latent reservoir. If antiretroviral therapy is stopped, the virus is able to resume replication and the disease progresses. Recently, it has been demonstrated that most of the latent reservoir capable of generating replication competent virus cannot be induced in the laboratory setting. However, the mechanisms that influence the generation of this intact and non-inducible latent reservoir are still under investigation. Here we demonstrate the generation of defective, intact and intact non-inducible latent HIV-1 in a TCM model of latency using different HIV-1 strains. Thus, the mechanisms which control inducibility can be studied using this primary cell model of latency, which may accelerate our understanding of the latent reservoir and the development of curative strategies.

Interactions with Commensal and Pathogenic Bacteria Induce HIV-1 Latency in Macrophages through Altered Transcription Factor Recruitment to the LTR.

Macrophages are infected by HIV-1 in vivo and contribute to both viral spread and pathogenesis. Recent human and animal studies suggest that HIV-1-infected macrophages serve as a reservoir that contributes to HIV-1 persistence during anti-retroviral therapy. The ability of macrophages to serve as persistent viral reservoirs is likely influenced by the local tissue microenvironment, including interactions with pathogenic and commensal microbes. Here we show that the sexually transmitted pathogen Neisseria gonorrhoeae (GC) and the gut-associated microbe Escherichia coli (E. coli), which encode ligands for both Toll-like receptor 2 (TLR2) and TLR4, repressed HIV-1 replication in macrophages and thereby induced a state reminiscent of viral latency. This repression was mediated by signaling through TLR4 and the adaptor protein TRIF and was associated with increased production of type I interferons. Inhibiting TLR4 signaling, blocking type 1 interferon, or knocking-down TRIF reversed LPS- and GC-mediated repression of HIV-1. Finally, the repression of HIV-1 in macrophages was associated with the recruitment of interferon regulatory factor 8 (IRF8) to the interferon stimulated response element (ISRE) downstream of the 5' HIV-1 long terminal repeat (LTR). Our data indicate that IRF8 is responsible for repression of HIV-1 replication in macrophages in response to TRIF-dependent signaling during GC and E. coli co-infection. These findings highlight the potential role of macrophages as HIV-1 reservoirs as well as the role of the tissue microenvironment and co-infections as modulators of HIV-1 persistence.IMPORTANCE The major barrier toward the eradication of HIV-1 infection is the presence of a small reservoir of latently infected cells, which include CD4+ T cells and macrophages that escape immune-mediated clearance and the effects of anti-retroviral therapy. There remain crucial gaps in our understanding of the molecular mechanisms that lead to transcriptionally silent or latent HIV-1 infection of macrophages. The significance of our research is in identifying microenvironmental factors, such as commensal and pathogenic microbes, that can contribute to the establishment and maintenance of latent HIV-1 infection in macrophages. It is hoped that identifying key processes contributing to HIV-1 persistence in macrophages may ultimately lead to novel therapeutics to eliminate latent HIV-1 reservoirs in vivo.

Screening for gene expression fluctuations reveals latency-promoting agents of HIV

Upon treatment removal, spontaneous reactivation of latently infected T cells remains a major barrier toward curing HIV. Therapies that reactivate and clear the latent reservoir are only partially effective, while latency-promoting agents (LPAs) used to suppress reactivation and stabilize latency are understudied and lack diversity in their mechanisms of action. Here, we identify additional LPAs using a screen for gene-expression fluctuations (or "noise") that drive cell-fate specification and control HIV reactivation from latency. Single-cell protein dynamics of a minimal HIV gene circuit were monitored with time-lapse fluorescence microscopy. We screened 1,806 drugs, out of which 279 modulate noise magnitude or half autocorrelation time. Next, we tested the strongest noise modulators in a Jurkat T cell latency model and discovered three LPAs that would be overlooked by quantifying their mean expression levels alone. The LPAs reduced reactivation of latency in both Jurkat and primary cell models when challenged by synergistic and potent combinations of HIV activators. The two strongest LPAs, NSC 401005 and NSC 400938, are structurally and functionally related to inhibitors of thioredoxin reductase, a protein involved in maintaining redox balance in host cells. Experiments with multiple functional analogs revealed two additional LPAs, PX12 and tiopronin, and suggest a potential LPA family, within which some are commercially available and Food and Drug Administration-approved. The LPAs presented here may provide new strategies to complement antiretroviral treatments. Screening for gene expression noise holds the potential for drug discovery in other diseases.

Phospholipid Metabolism Is Associated with Time to HIV Rebound upon Treatment Interruption.

ABSTRACTLipids are biologically active molecules involved in a variety of cellular processes and immunological functions, including inflammation. It was recently shown that phospholipids and their derivatives, lysophospholipids, can reactivate latent (dormant) tumor cells, causing cancer recurrence. However, the potential link between lipids and HIV latency, persistence, and viral rebound after cessation of antiretroviral therapy (ART) has never been investigated. We explored the links between plasma lipids and the burden of HIV during ART. We profiled the circulating lipidome from plasma samples from 24 chronically HIV-infected individuals on suppressive ART who subsequently underwent an analytic treatment interruption (ATI) without concurrent immunotherapies. The pre-ATI viral burden was estimated as time-to-viral-rebound and viral load set points post-ATI. We found that higher pre-ATI levels of lysophospholipids, including the proinflammatory lysophosphatidylcholine, were associated with faster time-to-viral-rebound and higher viral set points upon ART cessation. Furthermore, higher pre-ATI levels of the proinflammatory by-product of intestinal lysophosphatidylcholine metabolism, trimethylamine-N-oxide (TMAO), were also linked to faster viral rebound post-ART. Finally, pre-ATI levels of several phosphatidylcholine species (lysophosphatidylcholine precursors) correlated strongly with higher pre-ATI levels of HIV DNA in peripheral CD4+ T cells. Our proof-of-concept data point to phospholipids and lysophospholipids as plausible proinflammatory contributors to HIV persistence and rapid post-ART HIV rebound. The potential interplay between phospholipid metabolism and both the establishment and maintenance of HIV latent reservoirs during and after ART warrants further investigation.

Effect of the use of Galectin-9 and blockade of TIM-3 receptor in the latent cellular reservoir of HIV-1.

Reactivation of latent HIV-1 is a necessary step for the purging of the viral reservoir, although it does not seem to be enough. The stimulation of HIV-1 specific cytotoxic T lymphocytes (CTL) may be just as essential for this purpose. In this study, we aimed to show the effect of galectin-9 (Gal-9), known to revert HIV-1 latency, in combination with the blockade of TIM-3, a natural receptor for Gal-9 and an exhaustion marker. We confirmed the ability of Gal-9 to reactivate latent HIV-1 in Jurkat-LAT-GFP cells, as well as in an IL-7-based cellular model. This reactivation was not mediated via the TIM-3 receptor, but rather by the recognition of the Gal-9 of a specific oligosaccharide pattern of resting memory CD4+ T cells' surfaces. The potency of Gal-9 in inducing transcription of latent HIV-1 was equal to or greater than that of other latency-reversing agents (LRA). Furthermore, the combination of Gal-9 with other LRA did not show synergistic effects in the reactivation of the latent virus. To evaluate the impact of TIM-3 inhibition on the CTL-response, different co-culture experiments with CD4+T, CD8+ T, and NK cells were performed. Our data showed that blocking TIM-3 was associated with control of viral replication in both in vitro and ex vivo models in cells from PLWH on antiretroviral therapy. A joint strategy of the use of Gal-9 to reactivate latent HIV-1 and the inhibition of TIM-3 to enhance the HIV-1 CTL specific-response was associated with control of the replication of the virus that was being reactivated, thus potentially contributing to the elimination of the viral reservoir. Our results place this strategy as a promising approach to be tested in future studies. Reactivation of latent-HIV-1 by Gal-9 and reinvigoration of CD8+ T cells by TIM-3 blockade could be used separately or in combination.ImportanceHIV-1 infection is a health problem of enormous importance that still causes significant mortality. Antiretroviral treatment (ART) has demonstrated efficacy in the control of HIV-1 replication, decreasing the morbidity and mortality of the infection, but it cannot eradicate the virus. In our work, we tested a protein, galectin-9 (Gal-9), an HIV-1 latency-reversing agent, using an in vitro cellular model of latency and in cells from people living with HIV-1 (PLWH) on antiretroviral therapy. Our results confirmed the potential role of Gal-9 as a molecule with a potent HIV-1 reactivation capacity. More importantly, using a monoclonal antibody against T cell immunoglobulin and the mucin domain-containing molecule 3 (TIM-3) receptor we were able to enhance the HIV-1 cytotoxic T lymphocytes (CTL) specific response to eliminate the CD4+ T cells in which the virus had been reactivated. When used together, i.e., Gal-9 and TIM-3 blockade, control of the replication of HIV-1 was observed, suggesting a decrease in the cellular reservoir.

HIV-1 Productively Infects and Integrates in Bronchial Epithelial Cells.

BackgroundThe role of lung epithelial cells in HIV-1-related lung comorbidities remains unclear, and the major hurdle in curing HIV is the persistence of latent HIV reservoirs in people living with HIV (PLWH). The advent of combined antiretroviral therapy has considerably increased the life span; however, the incidence of chronic lung diseases is significantly higher among PLWH. Lung epithelial cells orchestrate the respiratory immune responses and whether these cells are productively infected by HIV-1 is debatable.MethodsNormal human bronchial epithelial cells (NHBEs) grown on air–liquid interface were infected with X4-tropic HIV-1LAV and examined for latency using latency-reversing agents (LRAs). The role of CD4 and CXCR4 HIV coreceptors in NHBEs were tested, and DNA sequencing analysis was used to analyze the genomic integration of HIV proviral genes, Alu-HIVgag-pol, HIV-nef, and HIV-LTR. Lung epithelial sections from HIV-infected humans and SHIV-infected macaques were analyzed by FISH for HIV-gag-pol RNA and epithelial cell-specific immunostaining.Results and DiscussionNHBEs express CD4 and CXCR4 at higher levels than A549 cells. NHBEs are infected with HIV-1 basolaterally, but not apically, by X4-tropic HIV-1LAV in a CXCR4/CD4-dependent manner leading to HIV-p24 antigen production; however, NHBEs are induced to express CCR5 by IL-13 treatment. In the presence of cART, HIV-1 induces latency and integration of HIV provirus in the cellular DNA, which is rescued by the LRAs (endotoxin/vorinostat). Furthermore, lung epithelial cells from HIV-infected humans and SHIV-infected macaques contain HIV-specific RNA transcripts. Thus, lung epithelial cells are targeted by HIV-1 and could serve as potential HIV reservoirs that may contribute to the respiratory comorbidities in PLWH.

Resveratrol Promotes HIV-1 Tat Accumulation via AKT/FOXO1 Signaling Axis and Potentiates Vorinostat to Antagonize HIV-1 Latency.

The latent reservoir of HIV-1 is a major barrier to achieving the eradication of HIV-1/AIDS. One strategy is termed "shock and kill", which aims to awaken the latent HIV-1 using latency reversing agents (LRAs) to replicate and produce HIV-1 particles. Subsequently, the host cells containing HIV-1 can be recognized and eliminated by the immune response and anti-retroviral therapy. Although many LRAs have been found and tested, their clinical trials were dissatisfactory. To aim of the study was to investigate how resveratrol reactivates silent HIV-1 transcription and assess if resveratrol could be a candidate drug for the "shock" phase in "shock and kill" strategy. We used established HIV-1 transcription cell models (HeLa-based NH1 and NH2 cells) and HIV-1 latent cell models (J-Lat A72 and Jurkat 2D10 cells). We performed resveratrol treatment on these cell lines and studied the mechanism of how resveratrol stimulates HIV-1 gene transcription. We also tested resveratrol's bioactivity on primary cells isolated from HIV-1 latent infected patients. Resveratrol promoted HIV-1 Tat protein levels, and resveratrol-induced Tat promotion was found to be dependent on the AKT/FOXO1 signaling axis. Resveratrol could partially dissociate P-TEFb (Positive Transcription Elongation Factor b) from 7SK snRNP (7SK small nuclear Ribonucleoprotein) and promote Tat-SEC (Super Elongation Complex) interaction. Preclinical studies showed that resveratrol potentiated Vorinostat to awaken HIV-1 latency in HIV-1 latent infected cells isolated from patients. We found a new mechanism of resveratrol stimulating the production of HIV-1. Resveratrol could be a promising candidate drug to eradicate HIV-1 reservoirs.

The Global Dynamics of HIV Latency Model Including Cell-to-Cell Viral Transmission

HIV spreads by cell-to-cell transfer and the release of cell-free particles. A slightly more effective method of retroviral transmission is the direct cell-to-cell transfer of HIV, according to recent reports. Intracellular interaction between unhealthy and healthy cells, in combination with cytokine discharged by the cells included, may affect the susceptibility of a target resting CD4+T cell to HIV infection and the formation of latent infection. We suggest a class of HIV latency mathematical model, integrating both cell-free virus transmission and direct cell-to-cell diffusion to improve the understanding of the dynamics of the latent reservoirs. We incorporate four components in our model: the uninfected T cells, the latently infected T cells, the active-infected T cells and the HIV viruses. We examine the latency model by introducing the basic reproduction number. We first establish the non-negativity and boundedness of the solutions of the system, and then we investigate the global stability of the steady states. The diseased-free equilibrium is globally stable when the basic reproduction number is less than 1 and if the basic reproduction number is greater than 1, the diseased equilibrium exists and is globally stable. Numerical simulations are executed to interpret the theoretical outcomes and evaluate the relative contribution of latency fractions in the virus production and the HIV latent reservoir by providing estimates.

Induction of Autophagy Reduces IFN-I Mediated Inflammation and Restores Anti-HIV-1 T Cell Response &lt;i&gt;in vivo&lt;/i&gt;

A hallmark of HIV-1 infection is chronic inflammation. Chronic immune activation drives the pathogenesis of HIV-1 infection, leading to loss of CD4+ T cells and exhaustion of antiviral cellular immunity. Previously, we demonstrated that persistent inflammation mediated by type I interferon (IFN-I) signaling drives T cell exhaustion. Combination of anti-retroviral therapy (ART) and IFN-I receptor blockade therapy led to accelerated viral suppression and reduced latent HIV-1 reservoirs, suggesting that targeting type I IFN signaling can be used as a therapeutic strategy to alleviate T cell exhaustion. However, as IFN-Is are key regulators for antiviral immunity, more specific interventions to fine-tune IFN-I signaling and chronic inflammation are needed. Autophagy is a homeostatic mechanism for disposal of damaged cellular organelles and elimination of intracellular pathogens, which is pivotal for cellular homeostasis, T cell development and function. Autophagy is also impaired during HIV-1 infection. Here we demonstrate that autophagy is directly linked to IFN-I signaling. Impairment of autophagy leads to accumulation of damaged mitochondria and spontaneous IFN-I signaling. Autophagy inducers reduce IFN-I signaling in activated macrophages and restore functions of exhausted anti-HIV-1 T cells in vitro. Importantly, autophagy inducer treatment in HIV-1 infected humanized bone marrow/liver/thymus (BLT) mice significantly reduced persistent IFN-I signaling and immune activation, restored exhausted antiviral T cell responses, and accelerated viral suppression by ART. Autophagy inducer treatment also led to reduced viral rebound after ART withdrawal. Taken together, our data suggest that therapeutically targeting autophagy could be used to treat persistent immune activation and improve immune control of HIV replication.