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 HIV reservoir) which decay slowly over time. Here, leveraging >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. 

Methods
Individuals with newly diagnosed acute (<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 (<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 <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 = 2x106 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 RPP30 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 >500 IPDA measurements, we interrogated a median of 4.8 x 105 CD4+ T cell genomes per assay and observed a median DNA shearing index (DSI) of 0.40.