Clonally expanded HIV-1 proviruses with 5'-leader defects can give rise to nonsuppressible residual viremia.

BackgroundAntiretroviral therapy (ART) halts HIV-1 replication, decreasing viremia to below the detection limit of clinical assays. However, some individuals experience persistent nonsuppressible viremia (NSV) originating from CD4+ T cell clones carrying infectious proviruses. Defective proviruses represent over 90% of all proviruses persisting during ART and can express viral genes, but whether they can cause NSV and complicate ART management is unknown.MethodsWe undertook an in-depth characterization of proviruses causing NSV in 4 study participants with optimal adherence and no drug resistance. We investigated the impact of the observed defects on 5'-leader RNA properties, virus infectivity, and gene expression. Integration-site specific assays were used to track these proviruses over time and among cell subsets.ResultsClones carrying proviruses with 5'-leader defects can cause persistent NSV up to approximately 103 copies/mL. These proviruses had small, often identical deletions or point mutations involving the major splicing donor (MSD) site and showed partially reduced RNA dimerization and nucleocapsid binding. Nevertheless, they were inducible and produced noninfectious virions containing viral RNA, but lacking envelope.ConclusionThese findings show that proviruses with 5'-leader defects in CD4+ T cell clones can give rise to NSV, affecting clinical care. Sequencing of the 5'-leader can help in understanding failure to completely suppress viremia.FundingOffice of the NIH Director and National Institute of Dental and Craniofacial Research, NIH; Howard Hughes Medical Institute; Johns Hopkins University Center for AIDS Research; National Institute for Allergy and Infectious Diseases (NIAID), NIH, to the PAVE, BEAT-HIV, and DARE Martin Delaney collaboratories.

Measuring the latent reservoir for HIV-1: Quantification bias in near full-length genome sequencing methods.

Antiretroviral therapy (ART) effectively inhibits HIV-1 replication but is not curative due to the persistence of a latent viral reservoir in resting CD4+ T cells. This reservoir is a major barrier to cure. Sequencing studies have revealed that the population of proviruses persisting in ART-treated individuals is dominated by defective proviruses that cannot give rise to viral rebound due to fatal defects including large deletions and APOBEC3-mediated hypermutation. Near full genome sequencing (nFGS) of individual proviruses is used in reservoir assays to provide an estimate of the fraction of proviruses that are intact. nFGS methods rely on a long-distance outer PCR capturing most (~9 kb) of the genome, followed by nested inner PCRs. The outer PCR is carried out at limit dilution, and interpretation of the results is based on the assumption that all proviruses are quantitatively captured. Here, we evaluate nFGS methods using the intact proviral DNA assay (IPDA), a multiplex digital droplet PCR assay that quantitates intact and defective proviruses with single molecule sensitivity using only short, highly efficient amplicons. We analyzed proviral templates of known sequence to avoid the additional complication of sequence polymorphism. With the IPDA, we quantitated molecular yields at each step of nFGS methods. We demonstrate that nFGS methods are inefficient and miss ~70% of full-length proviruses due to amplification failure at the initial outer PCR step. In contrast, proviruses with large internal deletions encompassing 70% of the genome can be quantitatively amplified under the same conditions. Accurate measurement of the latent reservoir of HIV-1 is essential for evaluating the efficacy of cure strategies, and the bias against full length proviruses in nFGS methods must be considered.

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.

Assessing the Suitability of Next-Generation Viral Outgrowth Assays to Measure Human Immunodeficiency Virus 1 Latent Reservoir Size.

BACKGROUND: Evaluations of human immunodeficiency virus (HIV) curative interventions require reliable and efficient quantification of replication-competent latent reservoirs. The "classic" quantitative viral outgrowth assay (QVOA) has been regarded as the reference standard, although prohibitively resource and labor intensive. We compared 6 "next-generation" viral outgrowth assays, using polymerase chain reaction or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA. METHODS: Next-generation QVOAs were compared with classic QVOA using single leukapheresis-derived samples from 5 antiretroviral therapy-suppressed HIV-infected participants and 1 HIV-uninfected control; each laboratory tested blinded batches of 3 frozen and 1 fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and laboratory levels. Models also estimated the effect of testing frozen versus fresh samples. RESULTS: Next-generation QVOAs had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher infectious units per million values than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95% credible interval [CI], 2.1-3.5-fold) for next-generation assays. Between-laboratory variation increased extra-Poisson variation to 3.4-fold (95% CI, 2.6-5.4-fold). Frozen storage did not substantially alter infectious units per million values (-18%; 95% CI, -52% to 39%). CONCLUSIONS: The data offer cautious support for use of next-generation QVOAs as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of latent reservoirs in eradication strategies would benefit from high throughput and scalable assays.

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.

Antigen-driven clonal selection shapes the persistence of HIV-1-infected CD4+ T cells in vivo.

Clonal expansion of infected CD4+ T cells is a major mechanism of HIV-1 persistence and a barrier to achieving a cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, and interaction with antigens. Here, we show that it is possible to link antigen responsiveness, the full proviral sequence, the integration site, and the T cell receptor ß-chain (TCRß) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated CMV- and Gag-responding CD4+ T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCRß repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), the proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCRß and integration site analysis showed that infection could occur early or late in the course of a clone's response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together, these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts.

Autologous IgG antibodies block outgrowth of a substantial but variable fraction of viruses in the latent reservoir for HIV-1.

In untreated HIV-1 infection, rapid viral evolution allows escape from immune responses. Viral replication can be blocked by antiretroviral therapy. However, HIV-1 persists in a latent reservoir in resting CD4+ T cells, and rebound viremia occurs following treatment interruption. The reservoir, which is maintained in part by clonal expansion, can be measured using quantitative viral outgrowth assays (QVOAs) in which latency is reversed with T cell activation to allow viral outgrowth. Recent studies have shown that viruses detected in QVOAs prior to treatment interruption often differ from rebound viruses. We hypothesized that autologous neutralizing antibodies directed at the HIV-1 envelope (Env) protein might block outgrowth of some reservoir viruses. We modified the QVOA to reflect pressure from low concentrations of autologous antibodies and showed that outgrowth of a substantial but variable fraction of reservoir viruses is blocked by autologous contemporaneous immunoglobulin G (IgG). A reduction in outgrowth of >80% was seen in 6 of 15 individuals. This effect was due to direct neutralization. We established a phylogenetic relationship between rebound viruses and viruses growing out in vitro in the presence of autologous antibodies. Some large infected cell clones detected by QVOA carried neutralization-sensitive viruses, providing a cogent explanation for differences between rebound virus and viruses detected in standard QVOAs. Measurement of the frequency of reservoir viruses capable of outgrowth in the presence of autologous IgG might allow more accurate prediction of time to viral rebound. Ultimately, therapeutic immunization targeting the subset of variants resistant to autologous IgG might contribute to a functional cure.

HSF1 inhibition attenuates HIV-1 latency reversal mediated by several candidate LRAs In Vitro and Ex Vivo.

HIV-1 latency is a major barrier to cure. Identification of small molecules that destabilize latency and allow immune clearance of infected cells could lead to treatment-free remission. In vitro models of HIV-1 latency involving cell lines or primary cells have been developed for characterization of HIV-1 latency and high-throughput screening for latency-reversing agents (LRAs). We have shown that the majority of LRAs identified to date are relatively ineffective in cells from infected individuals despite activity in model systems. We show here that, for diverse LRAs, latency reversal observed in model systems involves a heat shock factor 1 (HSF1)-mediated stress pathway. Small-molecule inhibition of HSF1 attenuated HIV-1 latency reversal by histone deactylase inhibitors, protein kinase C agonists, and proteasome inhibitors without interfering with the known mechanism of action of these LRAs. However, latency reversal by second mitochondria-derived activator of caspase (SMAC) mimetics was not affected by inhibition of HSF1. In cells from infected individuals, inhibition of HSF1 attenuated latency reversal by phorbol ester+ionomycin but not by anti-CD3+anti-CD28. HSF1 promotes elongation of HIV-1 RNA by recruiting P-TEFb to the HIV-1 long terminal repeat (LTR), and we show that inhibition of HSF1 attenuates the formation of elongated HIV-1 transcripts. We demonstrate that in vitro models of latency have higher levels of the P-TEFb subunit cyclin T1 than primary cells, which may explain why many LRAs are functional in model systems but relatively ineffective in primary cells. Together, these studies provide insights into why particular LRA combinations are effective in reversing latency in cells from infected individuals.

Single-cell transcriptional landscapes reveal HIV-1-driven aberrant host gene transcription as a potential therapeutic target.

Understanding HIV-1-host interactions can identify the cellular environment supporting HIV-1 reactivation and mechanisms of clonal expansion. We developed HIV-1 SortSeq to isolate rare HIV-1-infected cells from virally suppressed, HIV-1-infected individuals upon early latency reversal. Single-cell transcriptome analysis of HIV-1 SortSeq+ cells revealed enrichment of nonsense-mediated RNA decay and viral transcription pathways. HIV-1 SortSeq+ cells up-regulated cellular factors that can support HIV-1 transcription (IMPDH1 and JAK1) or promote cellular survival (IL2 and IKBKB). HIV-1-host RNA landscape analysis at the integration site revealed that HIV-1 drives high aberrant host gene transcription downstream, but not upstream, of the integration site through HIV-1-to-host aberrant splicing, in which HIV-1 RNA splices into the host RNA and aberrantly drives host RNA transcription. HIV-1-induced aberrant transcription was driven by the HIV-1 promoter as shown by CRISPR-dCas9-mediated HIV-1-specific activation and could be suppressed by CRISPR-dCas9-mediated inhibition of HIV-1 5' long terminal repeat. Overall, we identified cellular factors supporting HIV-1 reactivation and HIV-1-driven aberrant host gene transcription as potential therapeutic targets to disrupt HIV-1 persistence.

Differential decay of intact and defective proviral DNA in HIV-1-infected individuals on suppressive antiretroviral therapy.

BACKGROUNDThe relative stabilities of the intact and defective HIV genomes over time during effective antiretroviral therapy (ART) have not been fully characterized.METHODSWe used the intact proviral DNA assay (IPDA) to estimate the rate of change of intact and defective proviruses in HIV-infected adults on ART. We used linear spline models with a knot at seven years and a random intercept and slope up to the knot. We estimated the influence of covariates on rates of change.RESULTSWe studied 81 individuals for a median of 7.3 (IQR 5.9-9.6) years. Intact genomes declined more rapidly from initial suppression through seven years (15.7% per year decline; 95% CI -22.8%, -8.0%) and more slowly after seven years (3.6% per year; 95% CI -8.1%, +1.1%). The estimated half-life of the reservoir was 4.0 years (95% CI 2.7-8.3) until year seven and 18.7 years (95% CI 8.2-infinite) thereafter. There was substantial variability between individuals in the rate of decline until year seven. Intact provirus declined more rapidly than defective provirus (P < 0.001) and showed a faster decline in individuals with higher CD4+ T cell nadirs.CONCLUSIONThe biology of the replication-competent (intact) reservoir differs from that of the replication-incompetent (non-intact) pool of proviruses. The IPDA will likely be informative when investigating the impact of interventions targeting the reservoir.FUNDINGDelaney AIDS Research Enterprise, UCSF/Gladstone Institute of Virology & Immunology CFAR, CFAR Network of Integrated Systems, amfAR Institute for HIV Cure Research, I4C and Beat-HIV Collaboratories, Howard Hughes Medical Institute, Gilead Sciences, Bill and Melinda Gates Foundation.

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection.

HIV-1 latent reservoir size and diversity are stable following brief treatment interruption.

BACKGROUND: The effect of a brief analytical treatment interruption (ATI) on the HIV-1 latent reservoir of individuals who initiate antiretroviral therapy (ART) during chronic infection is unknown. METHODS: We evaluated the impact of transient viremia on the latent reservoir in participants who underwent an ATI and at least 6 months of subsequent viral suppression in a clinical trial testing the effect of passive infusion of the broadly neutralizing Ab VRC01 during ATI. RESULTS: Measures of total HIV-1 DNA, cell-associated RNA, and infectious units per million cells (IUPM) (measured by quantitative viral outgrowth assay [QVOA]) were not statistically different before or after ATI. Phylogenetic analyses of HIV-1 env sequences from QVOA and proviral DNA demonstrated little change in the composition of the virus populations comprising the pre- and post-ATI reservoir. Expanded clones were common in both QVOA and proviral DNA sequences. The frequency of clonal populations differed significantly between QVOA viruses, proviral DNA sequences, and the viruses that reactivated in vivo. CONCLUSIONS: The results indicate that transient viremia from ATI does not substantially alter measures of the latent reservoir, that clonal expansion is prevalent within the latent reservoir, and that characterization of latent viruses that can reactivate in vivo remains challenging. TRIAL REGISTRATION: ClinicalTrials.gov NCT02463227FUNDING. Funding was provided by the NIH.

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.

Defective HIV-1 Proviruses Are Expressed and Can Be Recognized by Cytotoxic T Lymphocytes, which Shape the Proviral Landscape.

Despite antiretroviral therapy, HIV-1 persists in memory CD4+ T cells, creating a barrier to cure. The majority of HIV-1 proviruses are defective and considered clinically irrelevant. Using cells from HIV-1-infected individuals and reconstructed patient-derived defective proviruses, we show that defective proviruses can be transcribed into RNAs that are spliced and translated. Proviruses with defective major splice donors (MSDs) can activate novel splice sites to produce HIV-1 transcripts, and cells with these proviruses can be recognized by HIV-1-specific cytotoxic T lymphocytes (CTLs). Further, cells with proviruses containing lethal mutations upstream of CTL epitopes can also be recognized by CTLs, potentially through aberrant translation. Thus, CTLs may change the landscape of HIV-1 proviruses by preferentially targeting cells with specific types of defective proviruses. Additionally, the expression of defective proviruses will need to be considered in the measurement of HIV-1 latency reversal.

Proliferation of latently infected CD4+ T cells carrying replication-competent HIV-1: Potential role in latent reservoir dynamics.

A latent reservoir for HIV-1 in resting CD4+ T lymphocytes precludes cure. Mechanisms underlying reservoir stability are unclear. Recent studies suggest an unexpected degree of infected cell proliferation in vivo. T cell activation drives proliferation but also reverses latency, resulting in productive infection that generally leads to cell death. In this study, we show that latently infected cells can proliferate in response to mitogens without producing virus, generating progeny cells that can release infectious virus. Thus, assays relying on one round of activation underestimate reservoir size. Sequencing of independent clonal isolates of replication-competent virus revealed that 57% had env sequences identical to other isolates from the same patient. Identity was confirmed by full-genome sequencing and was not attributable to limited viral diversity. Phylogenetic and statistical analysis suggested that identical sequences arose from in vivo proliferation of infected cells, rather than infection of multiple cells by a dominant viral species. The possibility that much of the reservoir arises by cell proliferation presents challenges to cure.