CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

558

Longitudinal Analysis of HIV Reservoir Dynamics Using Q4ddPCR in Individuals Who Are ART-Treated Rachel K. Scheck 1 , Gregory Gladkov 2 , Adam R. Ward 2 , Mark Melzer 1 , Deborah McMahon 3 , Ronald J. Bosch 4 , Bernard J. Macatangay 3 , Joshua C. Cyktor 3 , Joseph J. Eron 5 , John W. Mellors 3 , Rajesh T. Gandhi 6 , Lisa Buchauer 1 , R. Brad Jones 2 , Christian Gaebler 1 , for the ACTG A5321 Study Team 1 Charité – Universitätsmedizin Berlin, Berlin, Germany, 2 Weill Cornell Medicine, New York, NY, USA, 3 University of Pittsburgh, Pittsburgh, PA, USA, 4 Harvard TH Chan School of Public Health, Boston, MA, USA, 5 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 6 Harvard Medical School, Boston, MA, USA Background: Accurate, scalable methods for HIV reservoir quantification are crucial for cure studies. The Intact Proviral DNA Assay (IPDA) uses two targets in droplet digital PCR (ddPCR) and correctly identifies 90% of defective proviruses but can underestimate intact reservoir decline by misclassifying proviruses with genetic defects between target regions. Mathematical modeling predicts that adding more probes enhances accuracy in separating intact from defective proviruses. To address this, we developed Q4ddPCR, a high-throughput ddPCR, targeting 4 conserved regions in env, gag, packaging signal and pol, offering more specific quantification of intact HIV. Here, we describe reservoir characteristics of 42 PLWH using Q4ddPCR. Methods: We compared Q4ddPCR and IPDA readouts of 93 longitudinal samples from virally suppressed ACTG A5321 participants (32 participants with ≥ 2 and 18 with 3 timepoints in the first 4 years of ART): 9 female, 33 male sex (9 F, 32 M, 1 unknown gender) with median age 39 years and ART duration 1.8 years at baseline. Results: In participants with ≥2 timepoints, the majority showed reservoir decrease of ≥15% (24 by Q4ddPCR; 21 by IPDA). In some participants, reservoir increase or fluctuating reservoir size were observed. Over all longitudinal results, Q4ddPCR showed a larger decline in intact proviruses compared to IPDA (median 4.1-fold vs. 2.7-fold) over median 2.5 years (Fig.1A). The fraction of intact proviruses relative to total HIV-DNA was consistently lower in Q4ddPCR. Q4ddPCR revealed substantial differences in 72% of samples compared to IPDA, with a geometric mean ratio of 0.73 (range 0.2–1). Consistent with mathematical models predicting reduced specificity of 2-probe readouts after intact proviral decay, the greatest relative difference between both assays was observed at the latest timepoint (geometric mean ratio Q4ddPCR/IPDA 0.74 range 0.2-1; 0.74 range 0.3-1; 0.69 range 0.2-1 for timepoint 1, 2 and 3, respectively, Fig. 1B-D). While IPDA failed to quantify intact proviruses in 25% of samples, the use of additional and alternative probes in Q4ddPCR successfully rescued the quantification in 74% of these cases. Overall Q4ddPCR enabled the detection of intact proviral DNA in 94% of all samples. Conclusions: As predicted by modeling, Q4ddPCR reveals a smaller fraction of intact proviruses and a steeper decline compared to IPDA. These findings suggest that Q4ddPCR more accurately reflects intact reservoir dynamics, providing a specific tool for assessing HIV reservoirs. The figure, table, or graphic for this abstract has been removed. Paucity of HIV Envelope Expressing Cells Upon Latency Reversal in ART-Suppressed PWH Jonathan Richard 1 , Mehdi Benlarbi 2 , Remi Fromentin 1 , Lorie Marchitto 1 , Amélie Pagliuzza 1 , Amie Albertus 3 , Derek Yang 3 , Jean-Pierre Routy 4 , Amos B. Smith III 3 , Drew Weissman 3 , Edward Kreider 3 , Daniel E. Kaufmann 5 , Marzena Pazgier 6 , Nicolas Chomont 2 , Andrés Finzi 1 1 Centre de Recherche du CHUM, Montreal, Canada, 2 Université de Montréal, Montreal, Canada, 3 University of Pennsylvania, Philadelphia, PA, USA, 4 McGill University Health Centre Research Institute, Montreal, Canada, 5 University of Lausanne, Lausanne, Switzerland, 6 Uniformed Services University of the Health Sciences, Bethesda, MD, USA Background: Antiretroviral therapy (ART) effectively suppresses HIV-1 replication but fails to eradicate the virus, as HIV-1 persists in long-lived reservoirs. Therefore, novel strategies are needed to detect and eliminate them. The HIV-1 envelope glycoprotein (Env) is the only viral antigen expressed on the surface of infected cells, making it a prime target for immunotherapies. While current latency-reversing agents (LRAs) effectively induce p24 expression, their ability to stimulate Env expression remains unclear. Detecting Env on reactivated cells remains challenging. Broadly neutralizing antibodies (bnAbs) can recognize the native Env but so far failed to reliably detect it on reactivated cells. Some CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) recognize highly conserved epitopes exposed upon Env interaction with CD4 or CD4-mimetic compounds (CD4mc). Stemming from these nnAbs and CD4mc

properties and to inform future cure strategies, we developed Env-Flow, a novel flow cytometry-based assay designed to measure surface Env expression on CD4 T cells from PWH. Methods: We combined CD4i nnAbs (A32, 17b and 246D) with a potent CD4mc (CJF-III-288) to detect Env at the surface of CD4 T cells from 7 ART-treated and 6 untreated PWH. Env + cells were identified either as CD4 low Env + cells or using gp120 and gp41-specific CD4i nnAbs conjugated with distinct fluorochromes as Env +/+ cells. Alternatively, p24 expression was simultaneously measured using two anti-p24 mAbs (HIV-Flow). Results: The frequency of Env + cells in untreated PWH positively correlated with plasma viral load (p=0.02). In these viremic participants, the majority of p24 + cells co-expressed cell-surface Env (80.4% ± 13%). Sorted CD4 low Env + or Env +/+ cells from these participants confirmed the specificity of our assay as all Env+ cells harbored HIV-1 DNA. In ART-treated PWH, Env expression was observed upon reactivation with PMA/Ionomycin. Interestingly, only a fraction of reactivated p24+ cells displayed surface Env (36% ± 19.7%). These reactivated cells exhibited higher p24 levels and significantly lower surface Env expression compared to cells from viremic PWH, resulting in a reduced Env/p24 ratio (0.7 ± 0.5 vs 4 ± 2.3). Conclusions: Our findings highlight the scarcity of surface Env on reactivated cells from ART-treated PWH. Overall, Env-Flow offers a robust tool to identify and better characterize Env-expressing cells, as well as to evaluate LRAs for their ability to induce surface Env. Julie Janssens 1 , Cordelia M. Isbell 1 , Sun Jin Kim 1 , Alton Barbehenn 1 , Rebecca Hoh 1 , Michael Peluso 1 , Satish Pillai 2 , Nadia R. Roan 3 , Steven G. Deeks 1 , Sulggi Lee 1 , Steven Yukl 1 , Adam Wedrychowski 4 , Timothy J. Henrich 1 1 University of California San Francisco, San Francisco, CA, USA, 2 Vitalant Research Institute, San Francisco, CA, USA, 3 Gladstone Institutes, San Francisco, CA, USA, 4 San Francisco VA Medical Center, San Francisco, CA, USA Background: While early ART initiation has been associated with smaller HIV reservoirs, it is not clear how the timing of ART initiation affects subsets of the “active” HIV-transcribing reservoir. Methods: We compared temporal changes in various proviruses and HIV transcripts in CD4+T cells isolated from longitudinal blood samples obtained before ART (T1) and at two time points on suppressive ART (T2, T3) from 11 acute-treated PWH (median T2=119 days; T3=295 days) and 10 chronic-treated PWH (median T2=790 days; T3=1641 days). Initiated (TAR), 5’-elongated (R-U5 pre-Gag), mid-elongated (Pol), distal (Nef), completed (PolyA), and multiply spliced (Tat-Rev) HIV RNAs were measured by RT-ddPCR, normalized to cellular transcription and the corresponding HIV DNA levels, and expressed as the change in HIV levels divided by the change in time. Results: During untreated infection (T1), the acute cohort tended to have higher levels of some HIV DNA regions (P=0.02 for TAR; P=0.07 for Pol) and HIV transcripts (P=0.003 for Pol; median 1296 vs 56 copies/ug; P=0.07 for initiated and P=0.08 for completed RNA). After normalization to HIV DNA, however, the acute cohort showed no difference in elongated, Pol, or completed HIV RNA/ DNA and a trend toward lower initiated HIV RNA/DNA (P=0.08). By the first timepoint after ART suppression (T2), levels of multiply spliced HIV RNA were lower in the acute than chronic-treated PWH (P=0.012; median 0 vs. 2.8 copies/ ug), while we did not detect differences in other HIV RNA or RNA/DNA levels. From T1 to T2, the decrease in HIV per unit time was greater in the acute than chronic cohort for all HIV DNA regions (all P<0.05) and most HIV transcripts (all P<0.05, except P=0.07 for multiply spliced HIV RNA). The ratio of completed HIV RNA/DNA also tended to decay faster in the acute cohort (P=0.06), while no difference was observed for initiated, elongated, or Pol HIV RNA/DNA. Conclusions: Levels of TAR HIV DNA and Pol HIV RNA were higher in acute than chronic infection, but paradoxically, HIV transcription initiation tended to be lower in acute infection. After ART, acute-treated PWH showed faster declines in all HIV DNA regions and most HIV transcripts, as well as a trend towards faster decline in completed HIV RNA per provirus. These findings suggest that acute treatment preserves immune responses that are both better at killing infected cells and selectively reducing those that transcribe completed HIV transcripts. Early ART Initiation Is Associated With Faster Decay of the Transcriptionally Active HIV Reservoir

Poster Abstracts

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CROI 2025 147