DOI: 10.5061/dryad.q573n5tsd
title: Rapid biphasic decay of intact and defective HIV DNA reservoir during acute treated HIV disease

description: Despite antiretroviral therapy (ART), HIV persists in latently-infected
 cells (the HIV reservoir) which decay slowly over time. Here, leveraging
 &gt;500 longitudinal samples from 67 people living with HIV (PLWH)
 treated during acute infection, we developed a 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 PLWH. Defective DNA had a similar biphasic
 pattern, but data were more variable. Predicted intact and defective decay
 rates were faster for PLWH with earlier timing of ART initiation, higher
 initial CD4+ T cell count, and lower pre-ART viral load. In this study, we
 advanced our limited understanding of HIV reservoir decay at the time of
 ART initiation, informing future curative strategies targeting this
 critical time. 
description: Individuals with newly diagnosed acute (&lt;100 days) from
 HIV infection were enrolled in the UCSF Treat Acute HIV cohort and
 co-enrolled in the UCSF SCOPE HIV cohort, an ongoing longitudinal study of
 over 2,500 PWH. Eligible participants were provided same-day ART
 initiation with tenofovir/emtricitabine (TDF/FTC, then TAF/FTC once
 available in 2016) + dolutegravir (DTG) and linked to clinical care.
 Individuals reporting concomitant PrEP use (&lt;100 days from any
 potential exposure to HIV by history and/or clinical test results) were
 also started on darunavir+ritonavir (DRV/r) as a fourth drug, which was
 continued until confirmation of baseline HIV genotyping test results
 (Monogram Biosciences, South San Francisco, CA, U.S.A.). Additional ART
 changes necessary for clinical care (e.g., laboratory abnormalities,
 drug-drug interactions, and/or participant preference) were honored and
 adjusted during the period of study. Participants signed a release of
 information which allowed clinical data extraction to determine prior HIV
 negative test results from the SFDPH, as well as additional HIV test
 results. Study participants were seen for monthly study
 visits for the first 24 weeks (including an additional week 2 visit to
 confirm HIV test results from baseline visit) and then every 3-4 months
 thereafter. Inclusion criteria for the study were prior HIV negative
 testing within the last 90 days, laboratory-confirmed HIV-1 infection by
 antibody/antigen and/or plasma HIV RNA assay, and willingness to be
 participate in the study for at least 24 weeks. Participants with severe
 renal or hepatic impairment, concurrent treatment with immunomodulatory
 drugs, or exposure to any immunomodulatory drugs in the preceding 90 days
 prior to study entry, pregnant or breastfeeding women, or participants
 unwilling to agree to the use a double-barrier method of contraception
 throughout the study period, were excluded. For each study participant,
 the estimated date of detected HIV infection (EDDI) was calculated using
 the Infection Dating Tool (https://tools.incidence-estimation.org/idt/). At each visit, detailed interviews included questions regarding current medications, medication adherence, intercurrent illnesses, and hospitalizations were performed. In addition, peripheral blood sampling at each visit was performed to measure plasma HIV RNA (Abbott Real Time PCR assay, limit of detection &lt;40 copies/mL), CD4+ T cell count, and clinical labs (complete blood count, metabolic panel). All participants provided written informed consent, and the institutional review board of UCSF approved the research.  The frequencies of HIV intact and defective (3’ and 5’) DNA were quantified using the intact proviral DNA assay (IPDA). CD4+ T cells were isolated from cryopreserved PBMCs (EasySep Human CD4+ T cell Enrichment Kit, Stemcell Technologies), with cell count, viability, and purity assessed by flow cytometry. Negatively selected CD4+ T cells were recovered (median cells = 2x10<sup>6</sup> with median viability = 97%) and genomic DNA extracted using the QIAamp DNA Mini Kit (Qiagen). DNA concentration and quality were determined by fluorometry (Qubit dsDNA BR Assay Kit, Thermo Fisher Scientific) and ultraviolet-visible (UV/VIS) spectrophotometry (QIAxpert, Qiagen). The frequency of intact provirus was determined using two multiplex digital droplet polymerase chain reaction (ddPCR) assays performed in parallel: (1) the HIV-1 Proviral Discrimination reaction which distinguishes intact from defective provirus via two strategically placed amplicons in HIV psi and RRE regions as well as a hypermutation discrimination probe, and (2) the Copy Reference/Shearing reaction, which quantifies DNA shearing and input diploid cell equivalents using the human <em>RPP30</em> gene. All ddPCR reactions were assembled via automated liquid handles to maximize reproducibility and analyzed using the BioRad QX200 AutoDG Digital Droplet PCR system (BioRad). Up to 700 ng of genomic DNA were analyzed per reaction, and final input DNA concentrations were dependent upon recovered DNA concentrations. Samples were batch processed and analyzed, including negative controls from uninfected donors and J-Lat full-length clone 6.3 (E. Verdin, Gladstone Institutes and UCSF, San Francisco, CA, USA) cells as positive controls. Across &gt;500 IPDA measurements, we interrogated a median of 4.8 x 10<sup>5</sup> CD4+ T cell genomes per assay and observed a median DNA shearing index (DSI) of 0.40.
description: # Rapid Biphasic Decay of Intact and Defective HIV DNA Reservoir During
 Acute Treated HIV Disease
 [https://doi.org/10.5061/dryad.q573n5tsd](https://doi.org/10.5061/dryad.q573n5tsd) ## Description of the data and file structure Data were collected from individuals with newly diagnosed acute (&lt;100 days) from HIV infection were enrolled in the UCSF Treat Acute HIV cohort. Eligible participants were started on antiretroviral therapy (ART) and enrolled in our study. A total of 67 participants were enrolled. Study visits were scheduled at initial visit (week 0), results visit (week 2), and weeks 4, 8, 12, 16, 20, and 24. Baseline clinical measurements (CD4+ T cell count, HIV plasma viral load, estimated date of infection, and demographics) were taken at the initial visit. HIV plasma viral load (quantified with Abbott Real Time PCR assay, limit of detection &lt;40 copies/mL) and HIV DNA reservoir size (quantified with IPDA) were taken at each study visit.  ### Files and variables #### File: anonymized\_merged.csv **Description:**  ##### Variables * PID: Unique participant identifier * age_baseline: Participant age range at intake visit * ethnicity: Participant ethnicity (AA: African American , AS: Asian , LA: Latin American, WH: White, ZZ: Other) * eid_art_days: Estimated number of days between HIV acquisition and ART initiation * fiebig_keele: Fiebig stage (roman numeral denoting stage of untreated HIV) at time of ART initiation * prep_group: PrEP usage around time of HIV infection(0: no PrEP use, 1: HIV acquired on PrEP, 2: HIV acquisition/PrEP overlap &lt;10 days, 3: PrEP initiated but already acquired HIV) * first_cd4: CD4+ T cell count at initial visit * log10firstvl: log-10 plasma HIV viral load at initial visit * referral_site: Which HIV testing site referred the participant  * days_on_art: Days since ART initiation * assay: Which assay was run. HIV DNA is quantified using IPDA: Intact, Defective 3' (Defective3), Defective 5' (Defective5), total defective (Defective), and total provirus (Total_ProVirus). Plasma HIV viral load is denoted PlasmaVL. * CD4+ T Cell Viability: A measure of assay quality for IPDA results (higher is better). This measure is not relevant for the plasma HIV viral load; PlasmaVL is given a value of Inf by default to help thresholding. * concentration: The value of the assay. For IPDA measurements the concentration is in units of copies per million CD4+ T cells; for plasma HIV viral load the concentration is in units of copies/mL. ## Code/software No specific software is required to work with these plain-text csv data. Our data processing was done in R (4.3.1) using the tidyverse (2.0.0) library suite and mgcv (3.4.1). The analyses and visualization was done in R and is available in an R markdown file.