Replication-competent Proviruses Research Articles

The Hidden Enemy Within: Uncovering the Secrets of HIV Tissues Reservoirs and Current mRNA Vaccine Development.

Human Immunodeficiency Viruses (HIV) continue to pose a significant global health threat despite the availability of antiretroviral therapy (ART). As a retrovirus, HIV persists as a stable, integrated, and replication-competent provirus within a diverse array of long-lived cells for many years, often termed "latent reservoirs" in individuals. Thus, this review aims to furnish a comprehensive overview of diverse tissue reservoirs where HIV persists, elucidating their pathogenesis and advancement in their strategies for clinical management. Understanding the mechanisms underlying HIV persistence within tissue reservoirs is of significant interest in developing effective ART for suppressing the virus in the blood. In addition, we also discussed the ongoing mRNA HIV vaccine that has shown promising results in clinical trials to elicit broadly neutralizing antibodies and effective T-cell responses against HIV.

A novel high-throughput microwell outgrowth assay for HIV-infected cells.

Although antiretroviral therapy (ART) is effective at suppressing HIV replication, a viral reservoir persists that can reseed infection if ART is interrupted. Curing HIV will require elimination or containment of this reservoir, but the size of the HIV reservoir is highly variable between individuals. To evaluate the size of the HIV reservoir, several assays have been developed, including PCR-based assays for viral DNA, the intact proviral DNA assay, and the quantitative viral outgrowth assay (QVOA). QVOA is the gold standard assay for measuring inducible replication-competent proviruses, but this assay is technically challenging and time-consuming. To begin progress toward a more rapid and less laborious tool for quantifying cells infected with replication-competent HIV, we developed the Microwell Outgrowth Assay, in which infected CD4 T cells are co-cultured with an HIV-detecting reporter cell line in a polydimethylsiloxane (PDMS)/polystyrene array of nanoliter-sized wells. Transmission of HIV from infected cells to the reporter cell line induces fluorescent reporter protein expression that is detected by automated scanning across the array. Using this approach, we were able to detect HIV-infected cells from ART-naïve people with HIV (PWH) and from PWH on ART with large reservoirs. Furthermore, we demonstrate that infected cells can be recovered from individual rafts and used to analyze the diversity of viral sequences. Although additional development and optimization will be required for quantifying the reservoir in PWH with small latent reservoirs, this assay may be a useful prototype for microwell assays of infected cells.IMPORTANCEMeasuring the size of the HIV reservoir in people with HIV (PWH) will be important for determining the impact of HIV cure strategies. However, measuring this reservoir is challenging. We report a new method for quantifying HIV-infected cells that involves culturing cells from PWH in an array of microwells with a cell line that detects HIV infection. We show that this approach can detect rare HIV-infected cells and derive detailed virus sequence information for each infected cell.

HIV Expression in Infected T Cell Clones.

The principal barrier to an HIV-1 cure is the persistence of infected cells harboring replication-competent proviruses despite antiretroviral therapy (ART). HIV-1 transcriptional suppression, referred to as viral latency, is foremost among persistence determinants, as it allows infected cells to evade the cytopathic effects of virion production and killing by cytotoxic T lymphocytes (CTL) and other immune factors. HIV-1 persistence is also governed by cellular proliferation, an innate and essential capacity of CD4+ T cells that both sustains cell populations over time and enables a robust directed response to immunological threats. However, when HIV-1 infects CD4+ T cells, this capacity for proliferation can enable surreptitious HIV-1 propagation without the deleterious effects of viral gene expression in latently infected cells. Over time on ART, the HIV-1 reservoir is shaped by both persistence determinants, with selective forces most often favoring clonally expanded infected cell populations harboring transcriptionally quiescent proviruses. Moreover, if HIV latency is incomplete or sporadically reversed in clonal infected cell populations that are replenished faster than they are depleted, such populations could both persist indefinitely and contribute to low-level persistent viremia during ART and viremic rebound if treatment is withdrawn. In this review, select genetic, epigenetic, cellular, and immunological determinants of viral transcriptional suppression and clonal expansion of HIV-1 reservoir T cells, interdependencies among these determinants, and implications for HIV-1 persistence will be presented and discussed.

Intact proviruses are enriched in the colon and associated with PD-1+TIGIT− mucosal CD4+ T cells of people with HIV-1 on antiretroviral therapy

The persistence of intact replication-competent HIV-1 proviruses is responsible for the virological rebound off treatment. The gut could be a major reservoir of HIV-1 due to the high number of infected target cells. We collected blood samples and intestinal biopsies (duodenum, ileum, colon) from 42 people with HIV-1 receiving effective antiretroviral therapy. We used the Intact Proviral DNA Assay to estimate the frequency of intact HIV-1 proviruses in the blood and in the intestinal mucosa of these individuals. We analyzed the genetic complexity of the HIV-1 reservoir by performing single-molecule next-generation sequencing of HIV-1 env DNA. The activation/exhaustion profile of mucosal T lymphocytes was assessed by flow cytometry. Intact proviruses are particularly enriched in the colon. Residual HIV-1 transcription in the gut is associated with persistent mucosal and systemic immune activation. The HIV-1 intestinal reservoir appears to be shaped by the proliferation of provirus-hosting cells. The genetic complexity of the viral reservoir in the colon is positively associated with TIGIT expression but negatively with PD-1, and inversely related to its intact content. The size of the intact reservoir in the colon is associated with PD-1+TIGIT- mucosal CD4+ T cells, particularly in CD27+ memory cells, whose proliferation and survival could contribute to the enrichment of the viral reservoir by intact proviruses. Enrichment in intact proviruses makes the gut a key compartment for HIV-1 persistence on antiretroviral therapy. This project was supported by grants from the ANRS-MIE (ANRS EP61 GALT), Sidaction, and the Institut Universitaire de France.

The latent HIV reservoir: current advances in genetic sequencing approaches.

Multiple cellular HIV reservoirs in diverse anatomical sites can undergo clonal expansion and persist for years despite suppressive antiretroviral therapy, posing a major barrier toward an HIV cure. Commonly adopted assays to assess HIV reservoir size mainly consist of PCR-based measures of cell-associated total proviral DNA, intact proviruses and transcriptionally competent provirus (viral RNA), flow cytometry and microscopy-based methods to measure translationally competent provirus (viral protein), and quantitative viral outgrowth assay, the gold standard to measure replication-competent provirus; yet no assay alone can provide a comprehensive view of the total HIV reservoir or its dynamics. Furthermore, the detection of extant provirus by these measures does not preclude defects affecting replication competence. An accurate measure of the latent reservoir is essential for evaluating the efficacy of HIV cure strategies. Recent approaches have been developed, which generate proviral sequence data to create a more detailed profile of the latent reservoir. These sequencing approaches are valuable tools to understand the complex multicellular processes in a diverse range of tissues and cell types and have provided insights into the mechanisms of HIV establishment and persistence. These advancements over previous sequencing methods have allowed multiplexing and new assays have emerged, which can document transcriptional activity, chromosome accessibility, and in-depth cellular phenotypes harboring latent HIV, enabling the characterization of rare infected cells across restrictive sites such as the brain. In this manuscript, we provide a review of HIV sequencing-based assays adopted to address challenges in quantifying and characterizing the latent HIV reservoir.

AZD5582 plus SIV-specific antibodies reduce lymph node viral reservoirs in antiretroviral therapy-suppressed macaques

The main barrier to HIV cure is a persistent reservoir of latently infected CD4+ T cells harboring replication-competent provirus that fuels rebound viremia upon antiretroviral therapy (ART) interruption. A leading approach to target this reservoir involves agents that reactivate latent HIV proviruses followed by direct clearance of cells expressing induced viral antigens by immune effector cells and immunotherapeutics. We previously showed that AZD5582, an antagonist of inhibitor of apoptosis proteins and mimetic of the second mitochondrial-derived activator of caspases (IAPi/SMACm), induces systemic reversal of HIV/SIV latency but with no reduction in size of the viral reservoir. In this study, we investigated the effects of AZD5582 in combination with four SIV Env-specific Rhesus monoclonal antibodies (RhmAbs) ± N-803 (an IL-15 superagonist) in SIV-infected, ART-suppressed rhesus macaques. Here we confirm the efficacy of AZD5582 in inducing SIV reactivation, demonstrate enhancement of latency reversal when AZD5582 is used in combination with N-803 and show a reduction in total and replication-competent SIV-DNA in lymph-node-derived CD4+ T cells in macaques treated with AZD5582 + RhmAbs. Further exploration of this therapeutic approach may contribute to the goal of achieving an HIV cure.

The latent reservoir of inducible, infectious HIV-1 does not decrease despite decades of antiretroviral therapy.

HIV-1 persists in a latent reservoir in resting CD4+ T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first 7 years of ART (t1/2 = 44 months). However, whether decay continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After 7 years of ART, there was no long-term decrease in the frequency of inducible, replication-competent proviruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with t1/2 of 44 months did not continue with long-term ART. The lack of decay reflected proliferation of infected cells. Most inducible, replication-competent viruses (79.8%) had env sequences identical to those of other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4+ T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART.

Adaptation of the intact proviral DNA assay to a nanowell-based digital PCR platform

Quantification of intact proviruses is a critical measurement in HIV cure studies both in vitro and in vivo. The widely adopted ‘intact proviral DNA assay’ (IPDA), designed to discriminate and quantify genetically intact HIV proviruses based on detection of two HIV sequence-specific targets, was originally validated using Bio-Rad's droplet digital PCR technology (ddPCR). Despite its advantages, ddPCR is limited in multiplexing capability (two-channel) and is both labor- and time intensive. To overcome some of these limitations, we utilized a nanowell-based digital PCR platform (dPCR, QIAcuity from Qiagen) which is a fully automated system that partitions samples into nanowells rather than droplets. In this study we adapted the IPDA assay to the QIAcuity platform and assessed its performance relative to ddPCR. The dPCR could differentiate between intact, 5’ defective and 3’ defective proviruses and was sensitive to single HIV copy input. We found the intra-assay and inter-assay variability was within acceptable ranges (with coefficient of variation at or below 10%). When comparing the performance of the IPDA in ex vivo CD4+ T cells from people with HIV on antiretroviral therapy, there was a strong correlation in the quantification of intact (rs = 0.93; p < 0.001) and 3’ defective proviruses (rs = 0.96; p < 0.001) with a significant but less strong correlation for 5’ defective proviruses (rs = 0.7; p = 0.04). We demonstrate that the dPCR platform enables sensitive and accurate quantification of genetically intact and defective proviruses similar to the ddPCR system but with greater speed and efficiency. This flexible system can be further optimized in the future, to detect up to 5 targets, enabling a more precise detection of intact and potentially replication-competent proviruses.

HIV Reservoir: How to Measure It?

In the current quest for a complete cure for HIV/AIDS, the persistence of a long-lived reservoir of cells carrying replication-competent proviruses is the major challenge. Here, we describe the main elements and characteristics of several widely used assays of HIV latent reservoir detection. To date, researchers have developed several different HIV latent reservoir detection assays. Among them, the in vitro quantitative viral outgrowth assay (QVOA) has been the gold standard for assessing latent HIV-1 viral load. The intact proviral DNA assay (IPDA) based on PCR also demonstrated the predominance of defective viruses. However, these assays all have some drawbacks and may still be inadequate in detecting the presence of ultralow levels of latent virus in many patients who were initially thought to have been cured, but eventually showed viral rebound. An accurate and precise measurement of the HIV reservoir is therefore needed to evaluate curative strategies, aimed to functional cure or sterilizing cure.

Isolation of an Ecotropic Porcine Endogenous Retrovirus PERV-C from a Yucatan SLAD/D Inbred Miniature Swine.

Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

The Dynamic Linkage between Provirus Integration Sites and the Host Functional Genome Property Alongside HIV-1 Infections Associated with Antiretroviral Therapy.

(1) Background: The HIV-1 latent reservoir harboring replication-competent proviruses is the major barrier in the quest for an HIV-1 infection cure. HIV-1 infection at all stages of disease progression is associated with immune activation and dysfunctional production of proinflammatory soluble factors (cytokines and chemokines), and it is expected that during HIV-1 infection, different immune components and immune cells, in turn, participate in immune responses, subsequently activating downstream biological pathways. However, the functional interaction between HIV-1 integration and the activation of host biological pathways is presently not fully understood. (2) Methods: In this work, I used genes targeted by proviruses from published datasets to seek enriched immunologic signatures and host biological pathways alongside HIV-1 infections based on MSigDb and KEGG over-representation analysis. (3) Results: I observed that different combinations of immunologic signatures of immune cell types and proinflammatory soluble factors appeared alongside HIV-1 infections associated with antiretroviral therapy. Moreover, enriched KEGG pathways were often related to "cancer specific types", "immune system", "infectious disease viral", and "signal transduction". (4) Conclusions: The observations in this work suggest that the gene sets harboring provirus integration sites may define specific immune cells and proinflammatory soluble factors during HIV-1 infections associated with antiretroviral therapy.

Characterization of the Viral Reservoirs Among HIV-1 Non-B Vertically Infected Adolescents Receiving Antiretroviral Therapy: Protocol for an Observational and Comparative Study in Cameroon.

Antiretroviral therapy (ART) can bring HIV-1 levels in blood plasma to the undetectable level and allow a near-normal life expectancy for HIV-infected individuals. Unfortunately, ART is not curative and must be taken for life, because within a few weeks of treatment cessation, HIV viremia rebounds in most patients except for rare elite or posttreatment controllers of viremia. The primary source of this rebound is the highly stable reservoir of latent yet replication-competent HIV-1 proviruses integrated into the genomic DNA of the resting memory cluster of differentiation 4 (CD4+) T cells. To achieve a cure for HIV, understanding the cell reservoir environment is of paramount importance. The size and nature of the viral reservoir might vary according to the timing of therapy, therapeutic response, ART duration, and immune response. The mechanisms of reservoir maintenance generally depend on the levels/type of immune recognition; in addition, the dynamics of viral persistence are different between pediatric and adult populations. This difference could become more evident as children grow toward adolescence. We aim to characterize the HIV reservoirs and their variability as per the virological and immunological profiles of HIV-1 non-B vertically infected adolescents receiving ART in Cameroon during the Adolescents' Viral Reservoirs study to provide accurate and reliable data for HIV cure research. This study will involve HIV-1 non-B vertically infected adolescents selected from an existing cohort in our institution. Blood samples will be collected for analyzing immunological/virological profiles, including CD4/CD8 count, plasma viral load, immune activation/inflammatory markers, genotyping, and quantification of HIV-1 viral reservoirs. We will equally recruit an age-matched group of HIV-negative adolescents as control for immunological profiling. This study received funding in November 2021 and was approved by the national institutional review board in December 2021. Sample collection will start in November 2022, and the study will last for 18 months. The HIV-1 sequences generated will provide information on the circulating HIV-1 subtypes to guide the selection of the most appropriate ART for the participants. The levels of immune biomarkers will help determine the immune profile and help identify factors driving persistent immune activation/inflammation in HIV-infected adolescents compared to those in HIV-uninfected adolescents. Analysis of the virological and immunological parameters in addition to the HIV-1 reservoir size will shed light on the characteristics of the viral reservoir in adolescents with HIV-1 non-B infection. Our findings will help in advancing the knowledge on HIV reservoirs, in terms of size and genetic variability in adolescents living with HIV. Such evidence will also help in understanding the effects of ART timing and duration on the size of the reservoirs among adolescents living with HIV-a unique population from whom the findings generated will largely contribute to designing functional cure strategies. PRR1-10.2196/41473.

Distinct gene expression by expanded clones of quiescent memory CD4+ Tcells harboring intact latent HIV-1 proviruses.

SummaryAntiretroviral therapy controls, but does not cure, HIV-1 infection due to a reservoir of rare CD4+ T cells harboring latent proviruses. Little is known about the transcriptional program of latent cells. Here, we report a strategy to enrich clones of latent cells carrying intact, replication-competent HIV-1 proviruses from blood based on their expression of unique T cell receptors. Latent cell enrichment enabled single-cell transcriptomic analysis of 1,050 CD4+ T cells belonging to expanded clones harboring intact HIV-1 proviruses from 6 different individuals. The analysis reveals that most of these cells are T effector memory cells that are enriched for expression of HLA-DR, HLA-DP, CD74, CCL5, granzymes A and K, cystatin F, LYAR, and DUSP2. We conclude that expanded clones of latent cells carrying intact HIV-1 proviruses persist preferentially in a distinct CD4+ T cell population, opening possibilities for eradication.

HIV-1 latency is established preferentially in minimally activated and non-dividing cells during productive infection of primary CD4 T cells.

Latently infected CD4 T cells form a stable reservoir of HIV that leads to life-long viral persistence; the mechanisms involved in establishment of this latency are not well understood. Three scenarios have been proposed: 1) an activated, proliferating cell becomes infected and reverts back to a resting state; 2) an activated cell becomes infected during its return to resting; or 3) infection is established directly in a resting cell. The aim of this study was, therefore, to investigate the relationship between T cell activation and proliferation and the establishment of HIV latency. Isolated primary CD4 cells were infected at different time points before or after TCR-induced stimulation. Cell proliferation within acutely infected cultures was tracked using CFSE viable dye over 14 days; and cell subsets that underwent varying degrees of proliferation were isolated at end of culture by flow cytometric sorting. Recovered cell subpopulations were analyzed for the amount of integrated HIV DNA, and the ability to produce virus, upon a second round of cell stimulation. We show that cell cultures exposed to virus, prior to stimulus addition, contained the highest levels of integrated and replication-competent provirus after returning to quiescence; whereas, cells infected during the height of cell proliferation retained the least. Cells that did not divide or exhibited limited division, following virus exposure and stimulation contained greater amounts of integrated and inducible HIV than did cells that had divided many times. Based on these results, co-culture experiments were conducted to demonstrate that latent infection could be established directly in non-dividing cells via cell-to-cell transmission from autologous productively infected cells. Together, the findings from our studies implicate the likely importance of direct infection of sub-optimally activated T cells in establishment of latently infected reservoirs in vivo, especially in CD4 lymphocytes that surround productive viral foci within immune tissue microenvironments.

Cell-to-cell transmission of HIV-1 from provirus-activated cells to resting naïve and memory human primary CD4 T cells is highly efficient and requires CD4 and F-actin but not chemokine receptors.

Latently infected cells harboring replication‐competent proviruses represent a major barrier to HIV‐1 cure. One major effort to purge these cells has focused on developing the “shock and kill” approach for forcing provirus reactivation to induce cell killing by viral cytopathic effects, host immune responses, or both. We conducted kinetic and mechanistic studies of HIV‐1 protein expression, virion production, and cell‐to‐cell virus transmission during provirus reactivation. Provirus‐activated ACH‐2 cells stimulated with romidepsin (RMD) or PMA produced Nef early, and then Env and Gag in parallel with the appearance of virions. Env on the surface of provirus‐activated cells and cellular F‐actin were critical in the formation of virological synapses to mediate cell‐to‐cell transmission of HIV‐1 from provirus‐activated cells to uninfected cells. This HIV‐1 cell‐to‐cell transmission was substantially more efficient than transmission seen via cell‐free virus spread and required F‐actin remodeling and CD4, but not chemokine receptors. Resting human primary CD4+ T cells including naïve and memory subpopulations and, especially the memory CD4+ T cells, were highly susceptible to HIV‐1 infection via cell‐to‐cell transmission. Cell‐to‐cell transmission of HIV‐1 from provirus‐activated cells was profoundly decreased by protease inhibitors (PIs) and neutralizing antibodies (nAbs) that recognize the CD4‐binding site (CD4bs) such as VRC01, but not by reverse transcriptase (RT) inhibitor Emtricitabine (FTC). Therefore, our results suggest that PIs with potent blocking abilities should be used in clinical application of the “shock and kill” approach, most likely in combination with CD4bs nAbs, to prevent new HIV‐1 infections.

Toward a Functional Cure for HIV-1 Infection: The Block and Lock Therapeutic Approach

The persistence of latent, replication-competent HIV-1 proviruses in resting CD4+ T cells, and other cellular reservoirs, represents a major barrier to a cure. This reservoir is impervious to the immune system and to antiretroviral therapy, but has the potential to produce infectious rebound virus if antiretroviral therapy is interrupted. There are multiple ongoing efforts to identify and/or develop novel therapeutic strategies to eliminate or silence this latent reservoir of HIV-1 infection. One of these strategies is termed “block and lock”. The “block” refers to a therapeutic agent’s capacity to inhibit (or “block”) transcription of HIV-1 proviruses, while the “lock” refers to its capacity to induce permanent silencing of the proviruses, typically via repressive epigenetic modifications. The “block and lock” approach elicits a functional, rather than sterilizing, cure for HIV-1 infection. This review article focuses on therapeutic approaches (i.e., small molecules, nucleic acids and recombinant proteins) that have been identified to block and, in some cases, lock HIV-1 in the latent state. We also touch on critical research that needs to be accomplished to advance this approach into humans.

HibeRNAtion: HIV-1 RNA Metabolism and Viral Latency.

HIV-1 infection remains non-curative due to the latent reservoir, primarily a small pool of resting memory CD4+ T cells bearing replication-competent provirus. Pharmacological reversal of HIV-1 latency followed by intrinsic or extrinsic cell killing has been proposed as a promising strategy to target and eliminate HIV-1 viral reservoirs. Latency reversing agents have been extensively studied for their role in reactivating HIV-1 transcription in vivo, although no permanent reduction of the viral reservoir has been observed thus far. This is partly due to the complex nature of latency, which involves strict intrinsic regulation at multiple levels at transcription and RNA processing. Still, the molecular mechanisms that control HIV-1 latency establishment and maintenance have been almost exclusively studied in the context of chromatin remodeling, transcription initiation and elongation and most known LRAs target LTR-driven transcription by manipulating these. RNA metabolism is a largely understudies but critical mechanistic step in HIV-1 gene expression and latency. In this review we provide an update on current knowledge on the role of RNA processing mechanisms in viral gene expression and latency and speculate on the possible manipulation of these pathways as a therapeutic target for future cure studies.

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus-infected rhesus macaques on antiretroviral therapy.

Proliferation of latently infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to targeting this latent cell expansion is to inhibit mTOR, a regulatory kinase involved with cell growth, metabolism, and proliferation. Here, we determined the effects of chronic mTOR inhibition with rapamycin with or without T cell activation in SIV-infected rhesus macaques (RMs) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequency of proliferating CD4+ memory T cells (TM cells) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RMs and controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RMs on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin and CD3LALA–treated and control-treated RMs rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that, while rapamycin can decrease the proliferation of CD4+ TM cells, chronic mTOR inhibition alone or in combination with T cell activation was not sufficient to disrupt the stability of the SIV reservoir.

Annals for Educators - January 2022.

Annals for Educators - January 2022.

New Approaches to Multi-Parametric HIV-1 Genetics Using Multiple Displacement Amplification: Determining the What, How, and Where of the HIV-1 Reservoir.

Development of potential HIV-1 curative interventions requires accurate characterization of the proviral reservoir, defined as host-integrated viral DNA genomes that drive rebound of viremia upon halting ART (antiretroviral therapy). Evaluation of such interventions necessitates methods capable of pinpointing the rare, genetically intact, replication-competent proviruses within a background of defective proviruses. This evaluation can be achieved by identifying the distinct integration sites of intact proviruses within host genomes and monitoring the dynamics of these proviruses and host cell lineages over longitudinal sampling. Until recently, molecular genetic approaches at the single proviral level have been generally limited to one of a few metrics, such as proviral genome sequence/intactness, host-proviral integration site, or replication competency. New approaches, taking advantage of MDA (multiple displacement amplification) for WGA (whole genome amplification), have enabled multiparametric proviral characterization at the single-genome level, including proviral genome sequence, host-proviral integration site, and phenotypic characterization of the host cell lineage, such as CD4 memory subset and antigen specificity. In this review, we will examine the workflow of MDA-augmented molecular genetic approaches to study the HIV-1 reservoir, highlighting technical advantages and flexibility. We focus on a collection of recent studies in which investigators have used these approaches to comprehensively characterize intact and defective proviruses from donors on ART, investigate mechanisms of elite control, and define cell lineage identity and antigen specificity of infected CD4+ T cell clones. The highlighted studies exemplify how these approaches and their future iterations will be key in defining the targets and evaluating the impacts of HIV curative interventions.