CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

299LB RESURGENCE OF HIV-1 FOUNDER VIRUSES FOLLOWING ANTIRETROVIRAL TREATMENT INTERRUPTION Morgane Rolland 1 , Eric Sanders-Buell 1 , Meera Bose 1 , Nittaya Phanuphak 2 , Mark de Souza 3 , Nelson L. Michael 1 , Merlin L. Robb 1 , Jintanat Ananworanich 1 , Sodsai Tovanabutra 1 , for the RV254/SEARCH 010 Study Groups 1 US Military HIV Rsr Prog, Silver Spring, MD, USA, 2 Thai Red Cross AIDS Rsr Cntr, Bangkok, Thailand, 3 SEARCH, Thai Red Cross AIDS Rsr Cntr, Bangkok, Thailand Background: HIV-1 infected subjects, who started antiretroviral treatment in acute infection and were treated for several years, may be able to control HIV-1 replication following treatment interruption. Methods: Eight Thai participants (7 male, 1 female) started antiretroviral treatment days after HIV-1 diagnosis (Fiebig I) and participated in a treatment interruption study after more than 2 years of treatment (median: 1,005 days; range: 899-1994 days). HIV-1 pol sequences (1,791 nt) were obtained from plasma samples following the endpoint-dilution strategy. Results: Following a median of 26 days (range: 13-48) after treatment interruption, HIV-1 rebounded in all participants. The highest viral load in acute infection was a median of 9,358 copies/ml (range: 3,598-20,005). After treatment interruption, HIV-1 rebounded to a median of 38,254 copies/ml (range: 11,489-137,044) before treatment was re-initiated. We compared HIV-1 pol sequences amplified at a median of 3 days (range: 1-5) after HIV-1 diagnosis to sequences obtained a median of 6 days (range: 1-15) after HIV-1 rebound. Sequences from acute HIV-1 infection (n = 15) were used to infer the founder sequence. Most sequences (71%) at HIV-1 rebound were identical to the founder sequence: a median of 11 out of 15 (range: 9-13) sequences were identical, and 91% of sequences (a median of 14 out of 15; range: 12-15) had at most 1 mutation with the founder sequence. Across all participants, mutations were found as singletons unique to a given sequence except for one G-to-A transition that was shared across 3 sequences in one participant (P002) at HIV-1 rebound. There was no evidence of drug resistance mutations, nor any evidence of selection, either positive or negative, at HIV-1 rebound. There was also no evidence of HIV-1 evolution during the 2+ years of treatment as the sequences sampled at HIV-1 rebound were not more divergent from the founder than sequences sampled during acute HIV-1 infection (Mann Whitney test: 0.241< p <0.999). Conclusion: These results indicate that rebound HIV-1 resulted from the production of viral particles from latently infected CD4+ T cells (possibly clonally expanded during treatment) rather than from a continuous low level viral replication over the treatment years. These results demonstrate that antiretroviral treatment controls HIV-1 replication but is not sufficient to eliminate a viral reservoir that was established only for the first few days of HIV-1 infection.

Poster and Themed Discussion Abstracts

300 NOVEL ASSAY TO MEASURE INTEGRATED HIV DNA IN PBMC FROM ART-SUPPRESSED PERSONS Steven Lada 1 , Jake VanBelzan 2 , Caroline Ignacio 1 , Matthew Strain 1 , Una O’Doherty 2 , Douglas D. Richman 1 1 Univ of California San Diego, La Jolla, CA, USA, 2 Univ of Pennsylvania, Philadelphia, PA, USA

Background: Measuring integrated HIV DNA (provirus) in PBMC is important in characterizing the circulating HIV reservoir. However, these measures can be confounded by the presence of non-integrated linear and circular forms of HIV DNA. The Alu-Gag assay combined with an efficiency assumption is the current standard assay to measure integrated HIV DNA. Droplet digital PCR (ddPCR) provides an efficient, standard-independent method to measure HIV DNA, but unable to distinguish between integrated and unintegrated molecular forms. Previously, utilizing pulse-field gel electrophoresis (PFGE) we have shown a 98% clearance of circular forms of HIV DNA. Here, we applied PFGE to purify high- molecular weight DNA from linear and circular HIV DNA forms, and then assayed for integrated HIV DNA by ddPCR. In a blinded fashion, results of proviral levels were compared between Alu-Gag and PFGE+ddPCR assays. Methods: DNA was extracted from PBMC collected from 10 ART-suppressed persons. First, using between 2-63µg of extracted DNA, samples were quantified by a standard Alu-Gag assay. Second, 1µg of DNA was used to measure HIV 2-LTR, HIV Gag, and RPP30 without any prior separation by ddPCR. Third, 5µg of DNA from each sample was loaded per well of BluePippin Gel 0.75% DF Cassettes and separated using a PFGE 15kb High-Pass protocol, and levels of HIV 2-LTR, HIV Gag, and RPP30 were then measured by ddPCR. Results: Before PFGE separation, ddPCR detected episomal HIV 2-LTR in 6 samples and HIV Gag in all 10 samples. After processing with PFGE, HIV 2-LTR was undetectable while HIV Gag was still detected in all 10 samples. Cellular DNA recovery after PFGE, as measured by RPP30, varied between 9-42%with an average of 21% across all samples. Levels of integrated HIV DNA measured by Alu-Gag and PFGE proviral assays after unblinding were highly correlated (R=0.7051; p =0.023; Spearman’s Rank-Order Correlation). Conclusion: The PFGE+ddPCR Integrated HIV DNA assay removed episomal and linear species of HIV DNA to enable detection of provirus efficiently with as little as 5 μg (<500,000 cells) from HIV-infected persons receiving suppressive ART. These results were comparable to the standard Alu-Gag proviral assay, but the PFGE+ddPCR assay required fewer cells and was less technically difficult. Thus, the proposed PFGE+ddPCR assay provides a sensitive and precise approach to the measurement of integrated HIV DNA with sufficient throughput for translational research cure studies measuring the circulating HIV reservoir. 301 PARALLEL NEXTGEN FULL-LENGTH HIV PROVIRAL DNA SEQUENCE AND INTEGRATION SITE ANALYSIS Mathew L. Jones , Emanuele Marchi, Jacob Hurst, Nicola Robinson, John Frater Univ of Oxford, Oxford, UK Background: A small population of cells harbouring an integrated copy of HIV (‘the latent reservoir’) persist despite effective antiretroviral therapy (ART), and is the barrier to HIV cure. The rarity of these cells and absence of a unique cell-surface marker makes study of this population difficult. Additionally, proviral sequence analysis is complicated by the excess of human genomic material in the reaction. We present a novel DNA capture assay which combined with NextGen sequencing retrieves near full-length HIV sequences and integration sites from patient samples. Methods: Agilent’s SureSelect kit was modified to enrich for HIV proviral DNA. RNA baits homologous to HIV were hybridised to 3μg CD4 T-cell DNA for 20 hours. Enrichment was achieved by selection using streptavidin-coated beads. To improve specificity, wash steps were performed at 71°C. Captured DNA was sequenced using a MiSeq and reads mapped

CROI 2017 118