CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

the utility of using HIV-quantitative antibodies as a screening test for low circulating cell-associated HIV-1 DNA levels in children and adolescents with perinatal HIV-1 infection. Methods: This study utilized 514 longitudinally-collected plasma specimens from 61 perinatally-infected study participants living with HIV and enrolled in the Pediatric HIV/AIDS Cohort Study Adolescent Master Protocol (PHACS AMP). We included participants who achieved sustained virologic suppression (VS) with HIV-1 plasma levels <400 copies/mL at or before 5 years of age on ART and maintained virologic control (allowing for isolated viral loads ≥400 copies/ mL). Antibody levels to HIV-1 envelope (gp160, gp41), gag (capsid, p24; matrix, p17); RT (p66/51), and integrase (p31) were quantified by ELISA; PBMC HIV-1 DNA levels were measured by droplet digital PCR. Receiver operator curve (ROC) analyses and the random forest method were used to identify the most predictive antibodies for low HIV-1 DNA levels (<100 and <10 copies per million PBMCs). We also utilized ROC analysis to inform the stepwise building of a GEE model for low HIV-1 DNA levels that included all antibody levels as predictors. Results: Among the 13 children with VS by 1 year of age, antibodies to p17, p24, and RT decreased throughout follow-up and antibodies to gp160 and gp41 were low and remained low; antibodies to p31 were either exceedingly low or undetectable (Figure). In contrast, among the 48 children with late VS after 1 year of age (between 1-5 years), antibody levels to all six HIV-1 proteins were high and remained high or increased longitudinally. The stepwise model suggested that gp41 and gp160 were useful predictors of low HIV-1 DNA levels; c-statistics including all antibodies ranged from 0.75 to 0.77. The random forest method also identified gp41 and gp160 as important predictors of low HIV-1 DNA; area under the curve estimates using all HIV-1-specfic antibodies ranged from 0.70 to 0.81. Conclusion: HIV-1 antibody levels to gp41 and gp160 may be useful to identify virologically-suppressed children on ART with low circulating cell-associated HIV-1 DNA levels for inclusion in clinical trials aimed at remission.

311 A NOVEL DDPCR PROTOCOL TO ESTIMATE COPY NUMBERS OF POTENTIALLY INTACT HIV-1 PROVIRUS Claire Levy 1 , Sean Hughes 1 , Pavitra Roychoudhury 2 , Daniel B. Reeves 2 , Chelsea Amstuz 2 , Haiying Zhu 1 , Meei-Li Huang 1 , Yulun Wei 1 , Marta E. Bull 1 , Noah A. Cassidy 2 , Dara Lehman 2 , Jan McClure 1 , Robert Coombs 1 , Keith Jerome 2 , Florian Hladik 1 1 University of Washington, Seattle, WA, USA, 2 Fred Hutchinson Cancer Research Center, Seattle, WA, USA Background: Accurately quantifying the replication-competent HIV reservoir is essential for evaluating the efficacy of HIV cure strategies. Ideally, this should be achieved by a rapid turn-around high-throughput assay suitable for a clinical setting. Methods: We designed a multiplex ddPCR protocol to quantify potentially intact provirus in CD4+ T cells in ART-suppressed people living with HIV (PLWH). Our multiplex ddPCR targets 5 regions in the HIV genome across 2 ddPCR assays, each with 2 unique and 1 common target. We chose the 5 targets by selecting conserved sequences but with documented deletions from the LANL database. Multiplex ddPCR allows us to assess potentially intact (“intact”) proviral genomes by quantifying the number of droplets positive for all 3 targets. We developed a gentle DNA isolation method for cell and tissue samples, and also mathematically corrected for residual shearing, measured by two RPP30 targets. We normalized results to number of T cells, quantified by RPP30 (all cells) minus copies of a region in TRD that is deleted during TCR rearrangement and quantifies non-T cells. Results: Our method results in minimal shearing of DNA isolated from blood samples (mean: 90% un-sheared, SD: 6%), has a low limit of detection (96.1 copies/million T cells by probit analysis with 95% confidence), and high sensitivity (detection: 1-5 copies/million), specificity (100%, n=150 negative control tests) and reproducibility (CV of positive control aliquots tested 23x over 1-year: 42.8%). The final estimate of intact provirus is the lower of the 2 assays. In blood CD4+ T cells from 14 ART-suppressed PLWH, we measured HIV by QVOA (range: 0.08-3.49 infectious units/million) and ddPCR (0-1,900 copies/million, undetectable in 2/14 samples). ddPCR averaged 99.2x (range: 0-557x) higher than QVOA. Longitudinal CD4+ T cell samples from 6-8 blood draws over 4.5-10 years in 20 ART-suppressed PLWH showed median reservoir half-lives of 55 months (range: 22-∞), consistent with previous studies. To relate the mucosal tissue reservoir to HIV shedding, we tested 6 pairs of cervical biopsies (ddPCR) and vaginal secretions (HIV RNA). 3/6 were positive for intact provirus in tissues and viral RNA in secretions, 2/6 were negative for both, and 1/6 was positive by ddPCR but negative for viral RNA. Conclusion: Our protocol to quantify potentially intact HIV provirus is specific, sensitive, reproducible, and applicable to cell and tissue samples. 312 QUANTITATIVE HIV-1 SPECIFIC ANTIBODIES AS PREDICTORS OF BLOOD HIV-1 DNA LEVELS Margaret McManus 1 , Brad Karalius 2 , Kunjal Patel 2 , Deborah Persaud 3 , Katherine Luzuriaga 1 , for the Pediatric HIV/AIDS Cohort Study 1 University of Massachusetts, Worcester, MA, USA, 2 Harvard T.H. Chan School of Public Health, Boston, MA, USA, 3 Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Antiretroviral therapy (ART) reduces HIV-1-related morbidity and mortality in children but does not prevent the establishment of a persistent replication-competent HIV-1 reservoir. Achieving low reservoir size is favorable for HIV-1 eradication efforts and sustained virologic remission. We evaluated

Poster Abstracts

313LB LONGITUDINAL QVOA AND IPDA MEASUREMENTS IN CD4 T CELLS FROM ART-SUPPRESSED DONORS Shane Falcinelli 1 , Jenna Read 1 , Ross Murtagh 1 , Sam Raines 1 , Morgan Dewey 1 , Nancie Archin 1 , David M. Margolis 1 1 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Background: The intact proviral DNA assay (IPDA) is a novel method to quantify intact, latent provirus using minimal cell input relative to the gold standard quantitative viral outgrowth assay (QVOA). As IPDA sensitivity may be affected by viral diversity, prior to implementation in experimental medicine trials it is critical to evaluate the relationship between IPDA and QVOA measurements across different participants. As latent provirus can decay over time, a comparison of the IPDA to QVOA longitudinally is also needed. Methods: We conducted the IPDA on stored resting CD4 T cells from a cohort of 68 ART-suppressed individuals in whom QVOA had been measured. In 25 of these individuals, we performed the IPDA on two to six longitudinal samples, with matched QVOA data. Longitudinal sampling spanned a range of 1 to 33 years after ART initiation.

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