Single-cell epigenetic, transcriptional, and protein profiling of latent and active HIV-1 reservoir revealed that IKZF3 promotes HIV-1 persistence.

Understanding how HIV-1-infected cells proliferate and persist is key to HIV-1 eradication, but the heterogeneity and rarity of HIV-1-infected cells hamper mechanistic interrogations. Here, we used single-cell DOGMA-seq to simultaneously capture transcription factor accessibility, transcriptome, surface proteins, HIV-1 DNA, and HIV-1 RNA in memory CD4+ T cells from six people living with HIV-1 during viremia and after suppressive antiretroviral therapy. We identified increased transcription factor accessibility in latent HIV-1-infected cells (RORC) and transcriptionally active HIV-1-infected cells (interferon regulatory transcription factor [IRF] and activator protein 1 [AP-1]). A proliferation program (IKZF3, IL21, BIRC5, and MKI67 co-expression) promoted the survival of transcriptionally active HIV-1-infected cells. Both latent and transcriptionally active HIV-1-infected cells had increased IKZF3 (Aiolos) expression. Distinct epigenetic programs drove the heterogeneous cellular states of HIV-1-infected cells: IRF:activation, Eomes:cytotoxic effector differentiation, AP-1:migration, and cell death. Our study revealed the single-cell epigenetic, transcriptional, and protein states of latent and transcriptionally active HIV-1-infected cells and cellular programs promoting HIV-1 persistence.

Single-cell multiomics reveals persistence of HIV-1 in expanded cytotoxic T cell clones.

Understanding the drivers and markers of clonally expanding HIV-1-infected CD4+ T cells is essential for HIV-1 eradication. We used single-cell ECCITE-seq, which captures surface protein expression, cellular transcriptome, HIV-1 RNA, and TCR sequences within the same single cell to track clonal expansion dynamics in longitudinally archived samples from six HIV-1-infected individuals (during viremia and after suppressive antiretroviral therapy) and two uninfected individuals, in unstimulated conditions and after CMV and HIV-1 antigen stimulation. Despite antiretroviral therapy, persistent antigen and TNF responses shaped T cell clonal expansion. HIV-1 resided in Th1-polarized, antigen-responding T cells expressing BCL2 and SERPINB9 that may resist cell death. HIV-1 RNA+ T cell clones were larger in clone size, established during viremia, persistent after viral suppression, and enriched in GZMB+ cytotoxic effector memory Th1 cells. Targeting HIV-1-infected cytotoxic CD4+ T cells and drivers of clonal expansion provides another direction for HIV-1 eradication.

Biphasic decay of intact SHIV genomes following initiation of antiretroviral therapy complicates analysis of interventions targeting the reservoir.

The latent reservoir for HIV-1 in resting CD4+ T cells persists despite antiretroviral therapy (ART) and precludes cure. Reservoir-targeting interventions are evaluated in ART-treated macaques infected with simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV). Efficacy is determined by reservoir measurements before and after the intervention. However, most proviruses persisting in the setting of ART are defective. In addition, intact HIV-1 and SIV genomes undergo complex, multiphasic decay observable when new infection events are blocked by ART. Intervention-induced elimination of latently infected cells must be distinguished from natural decay. Here, we address these issues for SHIV. We describe an intact proviral DNA assay that allows digital counting of SHIV genomes lacking common fatal defects. We show that intact SHIV genomes in circulating CD4+ T cells undergo biphasic decay during the first year of ART, with a rapid first phase (t1/2 = 30.1 d) and a slower second phase (t1/2 = 8.1 mo) that is still more rapid that the slow decay observed in people with HIV-1 on long-term ART (t1/2 = 3.7 y). In SHIV models, most interventions are tested during 2nd phase decay. Natural 2nd phase decay must be considered in evaluating interventions as most infected cells present at this time do not become part of the stable reservoir. In addition, for interventions tested during 2nd phase decay, a caveat is that the intervention may not be equally effective in people with HIV on long-term ART whose reservoirs are dominated by latently infected cells with a slower decay rate.

Complex decay dynamics of HIV virions, intact and defective proviruses, and 2LTR circles following initiation of antiretroviral therapy.

In persons living with HIV-1 (PLWH) who start antiretroviral therapy (ART), plasma virus decays in a biphasic fashion to below the detection limit. The first phase reflects the short half-life (<1 d) of cells that produce most of the plasma virus. The second phase represents the slower turnover (t1/2 = 14 d) of another infected cell population, whose identity is unclear. Using the intact proviral DNA assay (IPDA) to distinguish intact and defective proviruses, we analyzed viral decay in 17 PLWH initiating ART. Circulating CD4+ T cells with intact proviruses include few of the rapidly decaying first-phase cells. Instead, this population initially decays more slowly (t1/2 = 12.9 d) in a process that largely represents death or exit from the circulation rather than transition to latency. This more protracted decay potentially allows for immune selection. After ∼3 mo, the decay slope changes, and CD4+ T cells with intact proviruses decay with a half-life of 19 mo, which is still shorter than that of the latently infected cells that persist on long-term ART. Two-long-terminal repeat (2LTR) circles decay with fast and slow phases paralleling intact proviruses, a finding that precludes their use as a simple marker of ongoing viral replication. Proviruses with defects at the 5' or 3' end of the genome show equivalent monophasic decay at rates that vary among individuals. Understanding these complex early decay processes is important for correct use of reservoir assays and may provide insights into properties of surviving cells that can constitute the stable latent reservoir.

TCR-mimic bispecific antibodies to target the HIV-1 reservoir.

HIV-1 infection is incurable due to the persistence of the virus in a latent reservoir of resting memory CD4+ T cells. "Shock-and-kill" approaches that seek to induce HIV-1 gene expression, protein production, and subsequent targeting by the host immune system have been unsuccessful due to a lack of effective latency-reversing agents (LRAs) and kill strategies. In an effort to develop reagents that could be used to promote killing of infected cells, we constructed T cell receptor (TCR)-mimic antibodies to HIV-1 peptide-major histocompatibility complexes (pMHC). Using phage display, we panned for phages expressing antibody-like variable sequences that bound HIV-1 pMHC generated using the common HLA-A*02:01 allele. We targeted three epitopes in Gag and reverse transcriptase identified and quantified via Poisson detection mass spectrometry from cells infected in vitro with a pseudotyped HIV-1 reporter virus (NL4.3 dEnv). Sequences isolated from phages that bound these pMHC were cloned into a single-chain diabody backbone (scDb) sequence, such that one fragment is specific for an HIV-1 pMHC and the other fragment binds to CD3ε, an essential signal transduction subunit of the TCR. Thus, these antibodies utilize the sensitivity of T cell signaling as readouts for antigen processing and as agents to promote killing of infected cells. Notably, these scDbs are exquisitely sensitive and specific for the peptide portion of the pMHC. Most importantly, one scDb caused killing of infected cells presenting a naturally processed target pMHC. This work lays the foundation for a novel therapeutic killing strategy toward elimination of the HIV-1 reservoir.

CRISPR-Cas attack of HIV-1 proviral DNA can cause unintended deletion of surrounding cellular DNA.

IMPORTANCE: Although HIV replication can be effectively inhibited by antiretroviral therapy, this does not result in a cure as the available drugs do not inactivate the integrated HIV-1 DNA in infected cells. Consequently, HIV-infected individuals need lifelong therapy to prevent viral rebound. Several preclinical studies indicate that CRISPR-Cas gene-editing systems can be used to achieve permanent inactivation of the viral DNA. It was previously shown that this inactivation was due to small inactivating mutations at the targeted sites in the HIV genome and to excision or inversion of the viral DNA fragment between two target sites. We, here, demonstrate that CRISPR-Cas treatment also causes large unintended deletions, which can include surrounding chromosomal sequences. As the loss of chromosomal sequences may cause oncogenic transformation of the cell, such unintended large deletions form a potential safety risk in clinical application of this antiviral application and possibly all CRISPR-Cas gene-editing approaches.

HIV-1 infection of genetically engineered iPSC-derived central nervous system-engrafted microglia in a humanized mouse model.

IMPORTANCE: Our mouse model is a powerful tool for investigating the genetic mechanisms governing central nervous system (CNS) human immunodeficiency virus type-1 (HIV-1) infection and latency in the CNS at a single-cell level. A major advantage of our model is that it uses induced pluripotent stem cell-derived microglia, which enables human genetics, including gene function and therapeutic gene manipulation, to be explored in vivo, which is more challenging to study with current hematopoietic stem cell-based models for neuroHIV. Our transgenic tracing of xenografted human cells will provide a quantitative medium to develop new molecular and epigenetic strategies for reducing the HIV-1 latent reservoir and to test the impact of therapeutic inflammation-targeting drug interventions on CNS HIV-1 latency.

Intestinal endothelial cells increase HIV infection and latency in resting and activated CD4 + T cells, particularly affecting CCR6 + CD4 + T cells.

BACKGROUND: With suppressive antiretroviral therapy, HIV infection is well-managed in most patients. However, eradication and cure are still beyond reach due to latent viral reservoirs in CD4 + T cells, particularly in lymphoid tissue environments including the gut associated lymphatic tissues. In HIV patients, there is extensive depletion of T helper cells, particularly T helper 17 cells from the intestinal mucosal area, and the gut is one of the largest viral reservoir sites. Endothelial cells line lymphatic and blood vessels and were found to promote HIV infection and latency in previous studies. In this study, we examined endothelial cells specific to the gut mucosal area-intestinal endothelial cells-for their impact on HIV infection and latency in T helper cells. RESULTS: We found that intestinal endothelial cells dramatically increased productive and latent HIV infection in resting CD4 + T helper cells. In activated CD4 + T cells, endothelial cells enabled the formation of latent infection in addition to the increase of productive infection. Endothelial-cell-mediated HIV infection was more prominent in memory T cells than naïve T cells, and it involved the cytokine IL-6 but did not involve the co-stimulatory molecule CD2. The CCR6 + T helper 17 subpopulation was particularly susceptible to such endothelial-cell-promoted infection. CONCLUSION: Endothelial cells, which are widely present in lymphoid tissues including the intestinal mucosal area and interact regularly with T cells physiologically, significantly increase HIV infection and latent reservoir formation in CD4 + T cells, particularly in CCR6 + T helper 17 cells. Our study highlighted the importance of endothelial cells and the lymphoid tissue environment in HIV pathology and persistence.

Inhibition of a Chromatin and Transcription Modulator, SLTM, Increases HIV-1 Reactivation Identified by a CRISPR Inhibition Screen.

Despite effective antiretroviral therapy, HIV-1 persistence in latent reservoirs remains a major obstacle to a cure. We postulate that HIV-1 silencing factors suppress HIV-1 reactivation and that inhibition of these factors will increase HIV-1 reactivation. To identify HIV-1 silencing factors, we conducted a genome-wide CRISPR inhibition (CRISPRi) screen using four CRISPRi-ready, HIV-1-d6-GFP-infected Jurkat T cell clones with distinct integration sites. We sorted cells with increased green fluorescent protein (GFP) expression and captured single guide RNAs (sgRNAs) via targeted deep sequencing. We identified 18 HIV-1 silencing factors that were significantly enriched in HIV-1-d6-GFPhigh cells. Among them, SLTM (scaffold attachment factor B-like transcription modulator) is an epigenetic and transcriptional modulator having both DNA and RNA binding capacities not previously known to affect HIV-1 transcription. Knocking down SLTM by CRISPRi significantly increased HIV-1-d6-GFP expression (by 1.9- to 4.2-fold) in three HIV-1-d6-GFP-Jurkat T cell clones. Furthermore, SLTM knockdown increased the chromatin accessibility of HIV-1 and the gene in which HIV-1 is integrated but not the housekeeping gene POLR2A. To test whether SLTM inhibition can reactivate HIV-1 and further induce cell death of HIV-1-infected cells ex vivo, we established a small interfering RNA (siRNA) knockdown method that reduced SLTM expression in CD4+ T cells from 10 antiretroviral therapy (ART)-treated, virally suppressed, HIV-1-infected individuals ex vivo. Using limiting dilution culture, we found that SLTM knockdown significantly reduced the frequency of HIV-1-infected cells harboring inducible HIV-1 by 62.2% (0.56/106 versus 1.48/106 CD4+ T cells [P = 0.029]). Overall, our study indicates that SLTM inhibition reactivates HIV-1 in vitro and induces cell death of HIV-1-infected cells ex vivo. Our study identified SLTM as a novel therapeutic target. IMPORTANCE HIV-1-infected cells, which can survive drug treatment and immune cell killing, prevent an HIV-1 cure. Immune recognition of infected cells requires HIV-1 protein expression; however, HIV-1 protein expression is limited in infected cells after long-term therapy. The ways in which the HIV-1 provirus is blocked from producing protein are unknown. We identified a new host protein that regulates HIV-1 gene expression. We also provided a new method of studying HIV-1-host factor interactions in cells from infected individuals. These improvements may enable future strategies to reactivate HIV-1 in infected individuals so that infected cells can be killed by immune cells, drug treatment, or the virus itself.

Layer-by-Layer Particles Deliver Epigenetic Silencing siRNA to HIV-1 Latent Reservoir Cell Types.

For over two decades, nanomaterials have been employed to facilitate intracellular delivery of small interfering RNA (siRNA), both in vitro and in vivo, to induce post-transcriptional gene silencing (PTGS) via RNA interference. Besides PTGS, siRNAs are also capable of transcriptional gene silencing (TGS) or epigenetic silencing, which targets the gene promoter in the nucleus and prevents transcription via repressive epigenetic modifications. However, silencing efficiency is hampered by poor intracellular and nuclear delivery. Here, polyarginine-terminated multilayered particles are reported as a versatile system for the delivery of TGS-inducing siRNA to potently suppress virus transcription in HIV-infected cells. siRNA is complexed with multilayered particles formed by layer-by-layer assembly of poly(styrenesulfonate) and poly(arginine) and incubated with HIV-infected cell types, including primary cells. Using deconvolution microscopy, uptake of fluorescently labeled siRNA is observed in the nuclei of HIV-1 infected cells. Viral RNA and protein are measured to confirm functional virus silencing from siRNA delivered using particles 16 days post-treatment. This work extends conventional particle-enabled PTGS siRNA delivery to the TGS pathway and paves the way for future studies on particle-delivered siRNA for efficient TGS of various diseases and infections, including HIV.

Sequence Evaluation and Comparative Analysis of Novel Assays for Intact Proviral HIV-1 DNA.

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size.IMPORTANCE The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART)-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Proviral DNA Circles with Reformed Long Terminal Repeats.

Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9. IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.

Low Inducibility of Latent Human Immunodeficiency Virus Type 1 Proviruses as a Major Barrier to Cure.

The latent reservoir for human immunodeficiency virus type 1 (HIV-1) in resting CD4+ T cells is a major barrier to cure. The dimensions of the reservoir problem can be defined with 2 assays. A definitive minimal estimate of the frequency of latently infected cells is provided by the quantitative viral outgrowth assay (QVOA), which detects cells that can be induced by T-cell activation to release infectious virus. In contrast, the intact proviral DNA assay (IPDA) detects all genetically intact proviruses and provides a more accurate upper limit on reservoir size than standard single-amplicon polymerase chain reaction assays which mainly detect defective proviruses. The frequency of cells capable of initiating viral rebound on interruption of antiretroviral therapy lies between the values produced by the QVOA and the IPDA. We argue here that the 1-2-log difference between QVOA and IPDA values in part reflects that the fact that many replication-competent proviruses are not readily induced by T-cell activation. Findings of earlier studies suggest that latently infected cells can be activated to proliferate in vivo without expressing viral genes. The proliferating cells nevertheless retain the ability to produce virus on subsequent stimulation. The low inducibility of latent proviruses is a major problem for the shock-and-kill strategy for curing HIV-1 infection, which uses latency-reversing agents to induce viral gene expression and render infected cells susceptible to immune clearance. The latency-reversing agents developed to date are much less effective at reversing latency than T-cell activation. Taken together, these results indicate that HIV-1 eradication will require the discovery of much more effective ways to induce viral gene expression.

Similar Frequency and Inducibility of Intact Human Immunodeficiency Virus-1 Proviruses in Blood and Lymph Nodes.

BACKGROUND: The human immunodeficiency virus (HIV)-1 latent reservoir (LR) in resting CD4+ T cells is a barrier to cure. LR measurements are commonly performed on blood samples and therefore may miss latently infected cells residing in tissues, including lymph nodes. METHODS: We determined the frequency of intact HIV-1 proviruses and proviral inducibility in matched peripheral blood (PB) and lymph node (LN) samples from 10 HIV-1-infected patients on antiretroviral therapy (ART) using the intact proviral DNA assay and a novel quantitative viral induction assay. Prominent viral sequences from induced viral RNA were characterized using a next-generation sequencing assay. RESULTS: The frequencies of CD4+ T cells with intact proviruses were not significantly different in PB versus LN (61/106 vs 104/106 CD4+ cells), and they were substantially lower than frequencies of CD4+ T cells with defective proviruses. The frequencies of CD4+ T cells induced to produce high levels of viral RNA were not significantly different in PB versus LN (4.3/106 vs 7.9/106), but they were 14-fold lower than the frequencies of cells with intact proviruses. Sequencing of HIV-1 RNA from induced proviruses revealed comparable sequences in paired PB and LN samples. CONCLUSIONS: These results further support the use of PB as an appropriate proxy for the HIV-1 LR in secondary lymphoid organs.

Nonstructured Treatment Interruptions Are Associated With Higher Human Immunodeficiency Virus Reservoir Size Measured by Intact Proviral DNA Assay in People Who Inject Drugs.

The latent reservoir for human immunodeficiency virus type 1 (HIV-1) in CD4+ T cells is a major barrier to cure. HIV-1-infected persons who inject drugs (PWID) often struggle to maintain suppression of viremia and experience nonstructured treatment interruptions (NTIs). The effects of injecting drugs or NTIs on the reservoir are unclear. Using the intact proviral DNA assay, we found no apparent effect of heroin or cocaine use on reservoir size. However, we found significantly larger reservoirs in those with frequent NTIs or a shorter interval from last detectable HIV RNA measurement. These results have important implications for inclusion of PWID in HIV-1 cure studies.

Relationship between intact HIV-1 proviruses in circulating CD4+ T cells and rebound viruses emerging during treatment interruption.

Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4+ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti-HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound.

Expanded cellular clones carrying replication-competent HIV-1 persist, wax, and wane.

The latent reservoir for HIV-1 in resting CD4+ T cells is a major barrier to cure. Several lines of evidence suggest that the latent reservoir is maintained through cellular proliferation. Analysis of this proliferative process is complicated by the fact that most infected cells carry defective proviruses. Additional complications are that stimuli that drive T cell proliferation can also induce virus production from latently infected cells and productively infected cells have a short in vivo half-life. In this ex vivo study, we show that latently infected cells containing replication-competent HIV-1 can proliferate in response to T cell receptor agonists or cytokines that are known to induce homeostatic proliferation and that this can occur without virus production. Some cells that have proliferated in response to these stimuli can survive for 7 d while retaining the ability to produce virus. This finding supports the hypothesis that both antigen-driven and cytokine-induced proliferation may contribute to the stability of the latent reservoir. Sequencing of replication-competent proviruses isolated from patients at different time points confirmed the presence of expanded clones and demonstrated that while some clones harboring replication-competent virus persist longitudinally on a scale of years, others wax and wane. A similar pattern is observed in longitudinal sampling of residual viremia in patients. The observed patterns are not consistent with a continuous, cell-autonomous, proliferative process related to the HIV-1 integration site. The fact that the latent reservoir can be maintained, in part, by cellular proliferation without viral reactivation poses challenges to cure.

Thiostrepton Reactivates Latent HIV-1 through the p-TEFb and NF-κB Pathways Mediated by Heat Shock Response.

Antiretroviral therapy (ART) suppresses HIV-1 replication but fails to cure the infection. The presence of an extremely stable viral latent reservoir, primarily in resting memory CD4+ T cells, remains a major obstacle to viral eradication. The "shock and kill" strategy targets these latently infected cells and boosts immune recognition and clearance, and thus, it is a promising approach for an HIV-1 functional cure. Although some latency-reversing agents (LRAs) have been reported, no apparent clinical progress has been made, so it is still vital to seek novel and effective LRAs. Here, we report that thiostrepton (TSR), a proteasome inhibitor, reactivates latent HIV-1 effectively in cellular models and in primary CD4+ T cells from ART-suppressed individuals ex vivo TSR does not induce global T cell activation, severe cytotoxicity, or CD8+ T cell dysfunction, making it a prospective LRA candidate. We also observed a significant synergistic effect of reactivation when TSR was combined with JQ1, prostratin, or bryostatin-1. Interestingly, six TSR analogues also show reactivation abilities that are similar to or more effective than that of TSR. We further verified that TSR upregulated expression of heat shock proteins (HSPs) in CD4+ T cells, which subsequently activated positive transcriptional elongation factor b (p-TEFb) and NF-κB signals, leading to viral reactivation. In summary, we identify TSR as a novel LRA which could have important significance for applications to an HIV-1 functional cure in the future.

Replicate Aptima Assay for Quantifying Residual Plasma Viremia in Individuals on Antiretroviral Therapy.

Detection of residual plasma viremia in antiretroviral therapy (ART)-suppressed HIV-infected individuals is critical for characterizing the latent reservoir and evaluating the impact of cure interventions. Ultracentrifugation-based single-copy assays are sensitive but labor intensive. Fully automated replicate testing using a standard clinical viral load assay was evaluated as a high-throughput alternative for the quantification of low-level viremia. Four plasma samples from blood donors with acute HIV-1 infection and one viral culture supernatant were serially diluted into 25-ml samples to nominal viral loads ranging from 39 to <0.5 copies (cp)/ml. Each dilution was tested with 45 replicates (reps) using 0.5 ml/rep with the Aptima HIV-1 Quant assay. The nominal and estimated viral loads based on the single-hit Poisson model were compared, and a hybrid Poisson digital model for calibrated viral load estimation was derived. Testing performed using 45 reps on longitudinal plasma samples from 50 ART-suppressed individuals in the Reservoir Assay Validation and Evaluation Network (RAVEN) study cohort (range of 1 to 19 years of continuous ART suppression) showed a median viral load of 0.54 cp/ml (interquartile range [IQR], 0.22 to 1.46 cp/ml) and a 14% (95% confidence interval [CI], 9% to 19%) decline in viral load for each additional year in duration suppressed. Within the RAVEN cohort, the expected false-negative rate for detection at lower rep numbers using 9 and 18 reps was 26% and 14%, respectively. Residual plasma viremia levels positively correlated with cell-associated HIV RNA and DNA. The performance characteristics of the replicate Aptima assay support its use for quantifying residual plasma viremia to study the latent HIV reservoir and cure interventions.

Expansion of Stem Cell-Like CD4+ Memory T Cells during Acute HIV-1 Infection Is Linked to Rapid Disease Progression.

Acute HIV-1 infection is characterized by high viremia and massive depletion of CD4+ T cells throughout all tissue compartments. During this time the latent viral reservoir is established but the dynamics of memory CD4+ T cell subset development, their infectability and influence on disease progression during acute HIV-1 infection has not been carefully described. We therefore investigated the dynamics of CD4+ T cell memory populations in the RV217 (ECHO) cohort during the acute phase of infection. Interestingly, while we found only small changes in central or effector memory compartments, we observed a profound expansion of stem cell-like memory CD4+ T cells (SCM) (2.7-fold; P < 0.0001). Furthermore, we demonstrated that the HIV-1 integration and replication preferentially take place in highly differentiated CD4+ T cells such as transitional memory (TM) and effector memory (EM) CD4+ T cells, while naive and less mature memory cells prove to be more resistant. Despite the relatively low frequency of productively infected SCM, we suggest that their quiescent phenotype, increased susceptibility to HIV-1 integration compared to naive cells and extensive expansion make them one of the key players in establishment and persistence of the HIV-1 reservoir. Moreover, the expansion of SCM in acute HIV-1 infection was a result of Fas upregulation on the surface of naive CD4+ T cells. Interestingly, the upregulation of Fas receptor and expansion of SCM in acute HIV-1 infection was associated with the early viral set point and disease progression (rho = 0.47, P = 0.02, and rho = 0.42, P = 0.041, respectively). Taken together, our data demonstrate an expansion of SCM during early acute HIV-1 infection which is associated with disease outcome.IMPORTANCE Understanding the immunopathology of acute HIV-1 infection will help to develop eradication strategies. We demonstrate here that a CD4+ T cell memory subset expands during acute HIV-1 infection, which is associated with disease progression.