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

Antiretroviral therapy (ART) is not a cure. Upon ART cessation, virus rapidly rebounds from latently-infected cells ("the HIV reservoir"). The reservoir is largely stabilized at the time of ART initiation and then decays slowly. 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 using the intact proviral DNA assay (IPDA) from peripheral CD4+ T cells. Nonlinear generalized additive models adjusted for initial CD4+ T count, pre-ART viral load, and timing of ART initiation demonstrated rapid biphasic decay of intact DNA (week 0-5: t1/2 ~0.71 months; week 5-24: t1/2 ~3.9 months) that extended out to 1 year of ART, with similar trends for defective DNA. Predicted reservoir decay were faster for participants individuals with earlier timing of ART initiation, higher initial CD4+ T cell count, and lower pre-ART viral load. These estimates are ~5-fold faster than prior reservoir decay estimates among chronic-treated PWH. Thus, these data add to our limited understanding of host viral control at the earliest stages of HIV reservoir stabilization, potentially informing future HIV cure efforts aimed at diverse, global population of PWH initiating ART at varying stages of disease.

Varied Patterns of Decay of Intact Human Immunodeficiency Virus Type 1 Proviruses Over 2 Decades of Antiretroviral Therapy.

Fourteen people with human immunodeficiency virus type 1 had longitudinal measurements of intact, defective, and total proviral DNA over the course of two decades of antiretroviral therapy. Three patterns of intact proviral DNA decay were revealed: (1) biphasic decline with markedly slower second-phase decline, (2) initial decline that transitions to a zero-slope plateau, and (3) initial decline followed by later increases in intact proviral DNA. Defective proviral DNA levels were essentially stable. Mechanisms of slowing or reversal of second-phase decay of intact proviral DNA may include the inability to clear cells with intact but transcriptionally silent proviruses and clonal expansion of cells with intact proviruses.

Single center, open label dose escalating trial evaluating once weekly oral ixazomib in ART-suppressed, HIV positive adults and effects on HIV reservoir size in vivo.

BACKGROUND: Achieving a functional or sterilizing cure for HIV will require identification of therapeutic interventions that reduce HIV reservoir size in infected individuals. Proteasome inhibitors, such as ixazomib, impact multiple aspects of HIV biology including latency, transcription initiation, viral replication, and infected cell killing through the HIV protease - Casp8p41 pathway, resulting in latency reversal and reduced measures of HIV reservoir size ex vivo. METHODS: We conducted a phase 1b/2a dose escalating, open label trial of weekly oral ixazomib for 24 weeks in antiretroviral (ART)-suppressed, HIV positive adults (NCT02946047). The study was conducted from March 2017 to August 2019 at two tertiary referral centers in the United States. The primary outcomes were safety and tolerability of oral ixazomib. Secondary outcomes included changes in immunologic markers and estimates of HIV reservoir size after ixazomib treatment. FINDINGS: Sixteen participants completed the study. Ixazomib up to 4mg weekly was safe and well-tolerated, yielding no treatment-emergent events above grade 1. In exploratory analyses, ixazomib treatment was associated with detectable viremia that was below the lower limit of quantification (LLQ) in 9 participants, and viremia that was above LLQ in 4 of 16 participants. While treatment was associated with reduced CD4 counts [baseline 783 cells/ mm3 vs. week-24 724 cells/ mm3 p=0.003], there were no changes in markers of cellular activation, exhaustion or inflammation. Total HIV DNA and proviral sequencing were not altered by ixazomib treatment. Intact proviral DNA assay (IPDA) identified intact proviruses in 14 patients pre-treatment, and in 10/14 of those subjects post treatment values were reduced (P=0.068), allowing a calculated intact proviral half life of 0.6 years (95% CI 0.3, 2.5), compared to 7.1 years (95% CI 3.9, 18, p=0.004) in historical controls. Differentiation Quantitative Viral Outgrowth Assays (dQVOA) identified measurable proviruses in 15 subjects pre-treatment; post-treatment values were numerically reduced in 9, but overall differences were not significantly different. INTERPRETATION: Our study successfully met its primary endpoint of demonstrating the safety of ixazomib for 24 weeks in HIV infected persons. Exploratory analyses suggest that the effects observed ex vivo of latency reversal and reductions in HIV reservoir size, also occur in vivo. Future controlled studies of ixazomib are warranted. FUNDING: This study was funded by Millennium Pharmaceuticals Inc..; the Mayo Clinic Foundation; the National Institutes of Health, including the National Institute of Allergy and Infectious Diseases, Division of AIDS, the National Heart, Lung and Blood Institute, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Neurological Disorders and Stroke, and the National Institute on Drug Abuse. Mayo Clinic also acknowledges generous funding support from Mr. Joseph T. and Mrs. Michele P. Betten.

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

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

Shared Mechanisms Govern HIV Transcriptional Suppression in Circulating CD103+ and Gut CD4+ T Cells.

Latent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor ß (TGF-ß), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-ß signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103+ and CD103- CD4 T cells from the blood and rectum of HIV-negative (HIV-) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV+) individuals. Like gut CD4+ T cells, circulating CD103+ cells harbored more HIV DNA than did CD103- cells but transcribed less HIV RNA per provirus. Circulating CD103+ cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103- cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103+ CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency.IMPORTANCE The ability of HIV to establish a reversibly silent, "latent" infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103+ CD4+ T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103+ T cells share a cellular transcriptome that more closely resembles that of CD4+ T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4+ or circulating CD103+ T cells from circulating CD103- T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV.

Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA.

A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1+ adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/106 CD4+ T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/106 CD4+ T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.

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.

Elite suppressors have low frequencies of intact HIV-1 proviral DNA.

: Elite controllers or suppressors control viral replication without antiretroviral therapy. We used the intact proviral DNA assay to approximate the size of the inducible latent reservoir in elite suppressors and found that, while the median frequency of both total and intact proviral DNA was markedly lower than the frequencies seen in chronic progressors on antiretroviral therapy there was no significant difference in the ratio of intact to total proviral DNA between elite suppressors and chronic progressors.