Intact Proviral DNA Assay Research Articles

Intact HIV Reservoir in Monocytes Is Associated With Cognitive Function in Virally Suppressed Women With HIV

Abstract Background Monocytes are susceptible to human immunodeficiency virus (HIV) infection, form HIV reservoirs, and contribute to central nervous system complications (eg, cognitive impairment) in virally suppressed women with HIV (vsWWH). However, it remains unknown if the quality and/or quantity of the monocyte reservoir contributes to cognition in vsWWH. Methods Sixty-two vsWWH (mean age = 56.1 years, SD = 7.1; 93% Black, non-Hispanic; all HIV RNA <250 copies/mL) completed a cognitive test battery, blood draw, and whole-blood immunophenotyping. Monocytes and CD4 T cells were isolated from a subset of 53 participants and the HIV reservoir was assessed using cell-specific intact proviral DNA assays (IPDA). Demographically adjusted z-scores were calculated for each outcome using data from participants without HIV in the Women's Interagency HIV Study. Cognitive outcomes of interest included domain-specific and global z-scores. Results Thirty-Eight percent of vsWWH had detectable intact HIV genomes in monocytes (median = 21.5 copies/million). Higher levels of intact HIV genomes per million monocytes were associated with poorer verbal memory (delayed recall, r = 0.55, P = .01; recognition, r = 0.46, P = .04), fine motor skills (r = 0.50, P = .03), and global function (r = 0.47, P = .04). Higher levels of intact HIV genomes in monocytes were associated with percent intermediate monocytes (r = 0.60, P = .008), and the ratio of intact per intermediate monocyte was associated with worse memory (r = −0.59, P = .008). There were no associations between CD4 reservoir and cognition. Conclusions The number of intact HIV genomes per million monocytes was related to poorer cognition and the percentage of intermediate monocytes. These findings suggest that the presence of HIV genomes in general do not relate to cognitive complications, but intact, and therefore potentially replication-competent HIV, may contribute to cognitive complications in vsWWH.

HIV-1-DNA/RNA and immunometabolism in monocytes: contribution to the chronic immune activation and inflammation in people with HIV-1

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

Galectin-9 Levels as a Potential Predictor of Intact HIV Reservoir Decay.

During antiretroviral therapy (ART), the HIV reservoir exhibits variability as cells with intact genomes decay faster than those with defective genomes, especially in the first years of therapy. The host factors influencing this decay are yet to be characterized. Observational study in 74 PWH on ART, of whom 70 (94.6%) were male. We used the intact proviral DNA assay to measure intact proviruses and Luminex immunoassay to measure 32 inflammatory cytokines in plasma. Linear spline models, with a knot at seven years, evaluated the impact of baseline cytokine levels and their trajectories on intact HIV kinetics over these years. Baseline Gal-9 was the most predictive marker for intact HIV kinetics, with lower Gal-9 predicting faster decay over the subsequent seven years. For each 10-fold decrease in Gal-9 at baseline, there was a mean 45% (95%CI 14%-84%) greater decay of intact HIV genomes per year. Conversely, higher baseline ITAC, IL-17, and MIP-1α predicted faster intact HIV decreases. Longitudinal changes in MIP-3α and IL-6 levels strongly associated with intact HIV kinetics, with a 10-fold increase in MIP-3α and a 10-fold decrease in IL-6 associated with a a 9.5% and 10% faster decay of intact HIV genomes per year, respectively. The pronounced association between baseline Gal-9 levels and subsequent intact HIV decay suggests that strategies reducing Gal-9 levels could accelerate reservoir decay. Additionally, the correlations of MIP-3α and IL-6 with HIV kinetics indicate a broader cytokine-mediated regulatory network, hinting at multi-targeted interventions that could modulate HIV reservoir dynamics.

Rapid Biphasic Decay of Intact and Defective HIV DNA Reservoir During Acute Treated HIV Disease.

Despite antiretroviral therapy (ART), HIV persists in latently-infected cells ("the reservoir") which decay slowly over time. Here, leveraging >500 longitudinal samples from 67 people with HIV (PWH) treated during acute infection, we developed a novel mathematical model to predict reservoir decay from peripheral CD4+ T cells. Nonlinear generalized additive models demonstrated rapid biphasic decay of intact DNA (week 0-5: t1/2~2.83 weeks; week 5-24: t1/2~15.4 weeks) that extended out to 1 year. These estimates were ~5-fold faster than prior decay estimates among chronic treated PWH. Defective DNA had a similar biphasic pattern, but data were more variable. Predicted intact and defective decay rates were faster for PWH with earlier timing of ART initiation, higher initial CD4+ T cell count, and lower pre-ART viral load. These data add to our limited understanding of HIV reservoir decay at the time of ART initiation, informing future curative strategies targeting this critical time.

HIV-1 subtype A1, D, and recombinant proviral genome landscapes during long-term suppressive therapy

The primary obstacle to curing HIV-1 is a reservoir of CD4+ cells that contain stably integrated provirus. Previous studies characterizing the proviral landscape, which have been predominantly conducted in males in the United States and Europe living with HIV-1 subtype B, have revealed that most proviruses that persist during antiretroviral therapy (ART) are defective. In contrast, less is known about proviral landscapes in females with non-B subtypes, which represents the largest group of individuals living with HIV-1. Here, we analyze genomic DNA from resting CD4+ T-cells from 16 female and seven male Ugandans with HIV-1 receiving suppressive ART (n = 23). We perform near-full-length proviral sequencing at limiting dilution to examine the proviral genetic landscape, yielding 607 HIV-1 subtype A1, D, and recombinant proviral sequences (mean 26/person). We observe that intact genomes are relatively rare and clonal expansion occurs in both intact and defective genomes. Our modification of the primers and probes of the Intact Proviral DNA Assay (IPDA), developed for subtype B, rescues intact provirus detection in Ugandan samples for which the original IPDA fails. This work will facilitate research on HIV-1 persistence and cure strategies in Africa, where the burden of HIV-1 is heaviest.

Machine learning approaches identify immunologic signatures of total and intact HIV DNA during long-term antiretroviral therapy.

Understanding the interplay between the HIV reservoir and the host immune system may yield insights into HIV persistence during antiretroviral therapy (ART) and inform strategies for a cure. Here, we applied machine learning approaches to cross-sectional high-parameter HIV reservoir and immunology data in order to characterize host-reservoir associations and generate new hypotheses about HIV reservoir biology. High-dimensional immunophenotyping, quantification of HIV-specific T cell responses, and measurement of genetically intact and total HIV proviral DNA frequencies were performed on peripheral blood samples from 115 people with HIV (PWH) on long-term ART. Analysis demonstrated that both intact and total proviral DNA frequencies were positively correlated with T cell activation and exhaustion. Years of ART and select bifunctional HIV-specific CD4 T cell responses were negatively correlated with the percentage of intact proviruses. A Leave-One-Covariate-Out (LOCO) inference approach identified specific HIV reservoir and clinical-demographic parameters, such as age and biological sex, that were particularly important in predicting immunophenotypes. Overall, immune parameters were more strongly associated with total HIV proviral frequencies than intact proviral frequencies. Uniquely, however, expression of the IL-7 receptor alpha chain (CD127) on CD4 T cells was more strongly correlated with the intact reservoir. Unsupervised dimension reduction analysis identified two main clusters of PWH with distinct immune and reservoir characteristics. Using reservoir correlates identified in these initial analyses, decision tree methods were employed to visualize relationships among multiple immune and clinical-demographic parameters and the HIV reservoir. Finally, using random splits of our data as training-test sets, machine learning algorithms predicted with approximately 70% accuracy whether a given participant had qualitatively high or low levels of total or intact HIV DNA. The techniques described here may be useful for assessing global patterns within the increasingly high-dimensional data used in HIV reservoir and other studies of complex biology.

The identification of intact HIV proviral DNA from human cerebrospinal fluid

We evaluated the HIV-1 DNA reservoir in peripheral blood mononuclear cells (PBMC) and cerebrospinal fluid (CSF) in people with HIV (PWH) and associations to cognitive dysfunction. Using the intact proviral DNA assay (IPDA), an emerging technique to identify provirus that may be the source of viral rebound, we assessed HIV DNA in CSF and PBMC in PWH regardless of antiretroviral therapy (ART). CSF was used as a sampling surrogate for the central nervous system (CNS) as opposed to tissue. IDPA results (3′ defective, 5′ defective, and intact HIV DNA) were analyzed by compartment (Wilcoxon signed rank; matched and unmatched pairs). Cognitive performance, measured via a battery of nine neuropsychological (NP) tests, were analyzed for correlation to HIV DNA (Spearman's rho). 11 CSF and 8 PBMC samples from PWH were evaluated both unmatched and matched. Total CSF HIV DNA was detectable in all participants and was significantly higher than in matched PBMCs (p ​= ​0.0039). Intact CSF HIV DNA was detected in 7/11 participants and correlated closely with those in PBMCs but tended to be higher in CSF than in PBMC. CSF HIV DNA did not correlate with global NP performance, but higher values did correlate with worse executive function (p ​= ​0.0440). Intact HIV DNA is frequently present in the CSF of PWH regardless of ART. This further supports the presence of an HIV CNS reservoir and provides a method to study CNS reservoirs during HIV cure studies. Larger studies are needed to evaluate relationships with CNS clinical outcomes.

Differential susceptibility of cells infected with defective and intact HIV proviruses to killing by obatoclax and other small molecules.

Some drugs that augment cell-intrinsic defenses or modulate cell death/survival pathways have been reported to selectively kill cells infected with HIV or Simian Immunodeficiency Virus (SIV), but comparative studies are lacking. We hypothesized that these drugs may differ in their ability to kill cells infected with intact and defective proviruses. To investigate this hypothesis, drugs were tested ex vivo on peripheral blood mononuclear cells (PBMC) from nine antiretroviral therapy (ART)-suppressed individuals. We tested drugs currently in clinical use or human trials, including auranofin (p53 modulator), interferon alpha2A, interferon gamma, acitretin (RIG-I inducer), GS-9620/vesatolimod (TLR7 agonist), nivolumab (PD-1 blocker), obatoclax (Bcl-2 inhibitor), birinapant [inhibitor of apoptosis proteins (IAP) inhibitor], bortezomib (proteasome inhibitor), and INK128/sapanisertib [mammalian target of rapamycin mTOR] [c]1/2 inhibitor). After 6 days of treatment, we measured cell counts/viabilities and quantified levels of total, intact, and defective HIV DNA by droplet digital PCR (Intact Proviral DNA Assay). Obatoclax reduced intact HIV DNA [median = 27-30% of dimethyl sulfoxide control (DMSO)] but not defective or total HIV DNA. Other drugs showed no statistically significant effects. Obatoclax and other Bcl-2 inhibitors deserve further study in combination therapies aimed at reducing the intact HIV reservoir in order to achieve a functional cure and/or reduce HIV-associated immune activation.

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-1 proviral DNA in purified peripheral blood CD34+ stem and progenitor cells in individuals with long-term HAART; paving the way to HIV gene therapy

Human immunodeficiency virus (HIV)-1 infection is an important public health problem worldwide. After primary HIV-1 infection, transcribed HIV-1 DNA is integrated into the host genome, serving as a reservoir of the virus and hindering a definite cure. Although highly active antiretroviral therapy suppresses active viral replication, resulting in undetectable levels of HIV RNA in the blood, a viral rebound can be detected after a few weeks of treatment interruption. This supports the concept that there is a stable HIV-1 reservoir in people living with HIV-1. Recently, a few individuals with HIV infection were reported to be probably cured by hematopoietic stem transplantation (HSCT). The underlying mechanism for this success involved transfusion of uninfected hematopoietic stem and progenitor cells (HSPCs) from CCR5-mutated donors who were naturally resistant to HIV infection. Thus, gene editing technology to provide HIV-resistant HSPC has promise in the treatment of HIV infections by HSCT. In this study, we aimed to find HIV-infected individuals likely to achieve a definite cure via gene editing HSCT. We screened for total HIV proviral DNA by Alu PCR in peripheral blood mononuclear cells (PBMCs) of 20 HIV-infected individuals with prolonged viral suppression. We assessed the amount of intact proviral DNA via a modified intact proviral DNA assay (IPDA) in purified peripheral CD34+ HSPCs. PBMCs from all 20 individuals were positive for the gag gene in Alu PCR, and peripheral CD34+ HSPCs were IPDA-negative for six individuals. Our results suggested that these six HIV-infected individuals could be candidates for further studies into the ability of gene editing HSCT to lead to a definite HIV cure.

Development of a highly sensitive and specific intact proviral DNA assay for HIV-1 subtype B and C

IntroductionHIV reservoir quantification is essential for evaluation of HIV curative strategies and may provide valuable insights about reservoir dynamics during antiretroviral therapy. The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C.MethodsPrimer and probe sequences were strategically positioned at conserved regions in psi and env and adapted to subtype B&C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict the specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability.ResultsThe in silico analysis showed that the HIV-1 B&C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (> 95% certainty) at 60 °C. Quantification of 2–6 copies can be performed with > 80% certainty. Lowering the annealing temperature to 55 °C slightly lowered the specificity but prevented exclusion of samples with single mutations in the primer/probe region.ConclusionsWe developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions.

Effects of COVID-19 mRNA vaccination on HIV viremia and reservoir size.

The immunogenic nature of coronavirus disease 2019 (COVID-19) mRNA vaccines led to some initial concern that these could stimulate the HIV reservoir. We analyzed changes in plasma HIV loads (pVL) and reservoir size following COVID-19 mRNA vaccination in 62 people with HIV (PWH) receiving antiretroviral therapy (ART), and analyzed province-wide trends in pVL before and after the mass vaccination campaign. Longitudinal observational cohort and province-wide analysis. Sixty-two participants were sampled prevaccination, and one month after their first and second COVID-19 immunizations. Vaccine-induced anti-SARS-CoV-2-Spike antibodies in serum were measured using the Roche Elecsys Anti-S assay. HIV reservoirs were quantified using the intact proviral DNA assay; pVL were measured using the cobas 6800 (lower limit of quantification: 20 copies/ml). The province-wide analysis included all 290 401 pVL performed in British Columbia, Canada between 2012 and 2022. Prevaccination, the median intact reservoir size was 77 [interquartile range (IQR): 20-204] HIV copies/million CD4 + T-cells, compared to 74 (IQR: 27-212) and 65 (IQR: 22-174) postfirst and -second dose, respectively (all comparisons P > 0.07). Prevaccination, 82% of participants had pVL <20 copies/ml (max: 110 copies/ml), compared to 79% postfirst dose (max: 183 copies/ml) and 85% postsecond dose (max: 79 copies/ml) ( P > 0.4). There was no evidence that the magnitude of the vaccine-elicited anti-SARS-CoV-2-Spike immune response influenced pVL nor changes in reservoir size ( P > 0.6). We found no evidence linking the COVID-19 mass vaccination campaign to population-level increases in detectable pVL frequency among all PWH in the province, nor among those who maintained pVL suppression on ART. We found no evidence that COVID-19 mRNA vaccines induced changes in HIV reservoir size nor plasma viremia.

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.

Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques.

The rebound-competent viral reservoir, composed of a virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after ART interruption (ATI), remains the biggest obstacle to treating HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop therapeutic strategies for reducing the rebound-competent viral reservoir. In this study, barcoded simian immunodeficiency virus (SIV), SIVmac239M, was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood and tissues from secondary lymphoid organs (spleen, mesenteric lymph nodes, and inguinal lymph nodes) and from the colon, ileum, lung, liver, and brain were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX and RNAscope in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy, although plasma viral RNA remained below 22 copies per milliliter. Among the tissues studied, mesenteric lymph nodes, inguinal lymph nodes, and spleen contained viral barcodes detected in plasma. CD4+ T cells were the main cell type harboring viral RNA after ATI. Furthermore, T cell zones in lymphoid tissues showed higher viral RNA abundance than B cell zones for most animals. These findings are consistent with lymphoid tissues contributing to the virus present in plasma early after ATI.

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.

Higher HIV-1 Env gp120-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) Activity Is Associated with Lower Levels of Defective HIV-1 Provirus.

A cure for HIV-1 (HIV) remains unrealized due to a reservoir of latently infected cells that persist during antiretroviral therapy (ART), with reservoir size associated with adverse health outcomes and inversely with time to viral rebound upon ART cessation. Once established during ART, the HIV reservoir decays minimally over time; thus, understanding factors that impact the size of the HIV reservoir near its establishment is key to improving the health of people living with HIV and for the development of novel cure strategies. Yet, to date, few correlates of HIV reservoir size have been identified, particularly in pediatric populations. Here, we employed a cross-subtype intact proviral DNA assay (CS-IPDA) to quantify HIV provirus between one- and two-years post-ART initiation in a cohort of Kenyan children (n = 72), which had a median of 99 intact (range: 0-2469), 1340 defective (range: 172-3.84 × 104), and 1729 total (range: 178-5.11 × 104) HIV proviral copies per one million T cells. Additionally, pre-ART plasma was tested for HIV Env-specific antibody-dependent cellular cytotoxicity (ADCC) activity. We found that pre-ART gp120-specific ADCC activity inversely correlated with defective provirus levels (n = 68, r = -0.285, p = 0.0214) but not the intact reservoir (n = 68, r = -0.0321, p-value = 0.800). Pre-ART gp41-specific ADCC did not significantly correlate with either proviral population (n = 68; intact: r = -0.0512, p-value = 0.686; defective: r = -0.109, p-value = 0.389). This suggests specific host immune factors prior to ART initiation can impact proviruses that persist during ART.

CD8+ cells and small viral reservoirs facilitate post-ART control of SIV replication in M3+ Mauritian cynomolgus macaques initiated on ART two weeks post-infection.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). None of the MCMs possessed MHC haplotypes previously associated with SIV control. For six months after ART withdrawal, we observed undetectable or transient viremia in seven of the eight MCMs, despite detecting replication competent SIV using quantitative viral outgrowth assays. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the observed PTC was mediated, at least in part, by CD8α+ cells. With intact proviral DNA assays, we found that MCMs had significantly smaller viral reservoirs two wpi than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. We found a similarly small viral reservoir among six additional SIV+ MCMs in which ART was initiated at eight wpi, some of whom exhibited viral rebound. These results suggest that an unusually small viral reservoir is a hallmark among SIV+ MCMs. By evaluating immunological differences between MCMs that did and did not rebound, we identified that PTC was associated with a reduced frequency of CD4+ and CD8+ lymphocyte subsets expressing exhaustion markers. Together, these results suggest a combination of small reservoirs and immune-mediated virus suppression contribute to PTC in MCMs. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.

Virological and immunological correlates of HIV post-treatment control after temporal antiretroviral therapy during acute HIV infection.

People with HIV rarely control viral replication after cessation of antiretroviral therapy (ART). We present a person with HIV with extraordinary post-treatment control (PTC) for over 23 years after temporary ART during acute HIV infection (AHI) leading to a new insight in factors contributing to PTC. Viral reservoir was determined by HIV qPCR, Intact Proviral DNA Assay and quantitative viral outgrowth assay. Viral replication kinetics were determined in autologous and donor PBMC. IgG levels directed against HIV envelope and neutralizing antibodies were measured. Immune phenotyping of T-cells and HIV-specific T-cell responses were analyzed by flow cytometry. The case presented with AHI and a plasma viral load of 2.7 million copies/mL. ART was initiated 2 weeks after diagnosis and interrupted after 26 months. Replicating virus was isolated shortly after start ART. At 18 years after treatment interruption HIV-DNA in CD4+ T-cells and low levels of HIV-RNA in plasma (<5 copies/ml) were detectable. Stable HIV envelope glycoprotein-directed IgG was present during follow-up, but lacked neutralizing activity. Strong antiviral CD8+ T-cell responses, in particular targeting HIV-gag, were detected during 25 years follow-up. Moreover, we found a P255A mutation in an HLA-B∗44:02 restricted gag-epitope, which was associated with decreased replication. We describe an exceptional case of post-treatment control, which is likely associated with sustained potent gag-specific CD8+ T-cell responses in combination with a replication attenuating escape mutation in gag. Understanding the initiation and preservation of the HIV-specific T-cell responses could guide the development of strategies to induce HIV control.

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.

Impact of misclassified defective proviruses on HIV reservoir measurements

Most proviruses persisting in people living with HIV (PWH) on antiretroviral therapy (ART) are defective. However, rarer intact proviruses almost always reinitiate viral rebound if ART stops. Therefore, assessing therapies to prevent viral rebound hinges on specifically quantifying intact proviruses. We evaluated the same samples from 10 male PWH on ART using the two-probe intact proviral DNA assay (IPDA) and near full length (nfl) Q4PCR. Both assays admitted similar ratios of intact to total HIV DNA, but IPDA found ~40-fold more intact proviruses. Neither assay suggested defective proviruses decay over 10 years. However, the mean intact half-lives were different: 108 months for IPDA and 65 months for Q4PCR. To reconcile this difference, we modeled additional longitudinal IPDA data and showed that decelerating intact decay could arise from very long-lived intact proviruses and/or misclassified defective proviruses: slowly decaying defective proviruses that are intact in IPDA probe locations (estimated up to 5%, in agreement with sequence library based predictions). The model also demonstrates how misclassification can lead to underestimated efficacy of therapies that exclusively reduce intact proviruses. We conclude that sensitive multi-probe assays combined with specific nfl-verified assays would be optimal to document absolute and changing levels of intact HIV proviruses.