CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

Background: Plasma HIV-1 RNA above the limit of detection of commercial assays on ART arise from i)complete cycles of viral replication as a consequence of inadequate drug exposure and/or drug resistance; or from ii)virions produced from proviruses in clonally-expanded cells without complete cycles of replication. Most proviruses that persist on ART are defective, including those hypermutated by APOBEC. Although hypermutated proviruses can be transcribed into mRNA and even spliced, their packaging into virions is expected to be very inefficient. Here we report the first instance of false virologic failure on ART arising from cells with hypermutated proviruses. Methods: A 46 year old female presented with detectable HIV on ART ranging from 439 to 4230 copies/mL. Single genome sequencing (SGS) analysis (p6-Pro- RT) of 4 longitudinal plasma samples obtained over 13 months was performed. To characterize the source of viremia, fractions of plasma after low- (2700 g) and high-speed centrifugation (17 200 g) and total nucleic acid from PBMC were analyzed by SGS including cell-associated HIV mRNA and near-full length (NFL) sequencing of proviral DNA. Results: SGS (p6-Pro-RT) revealed multiple, identical hypermutated sequences in all low-and one high-speed plasma pellet(s), and in PBMC HIV DNA (p6-Pro- RT and NFL) and cell-associated HIV mRNA (Figure). The only non-hypermutated sequences were from the high-speed plasma pellet (4 of 4) and PBMC HIV DNA (1 of 10) at the 13 month time point. Sequencing of gag revealed a stop codon at amino acid 211 which would prevent capsid (p24) formation. Conclusion: This is the first report of false virologic failure of ART resulting from release of defective HIV nucleic acid into plasma. The source appears to be a large population of cells with identical hypermutated proviruses, i.e. an infected CD4+ T-cell clone that is undergoing cytolysis and release of cellular nucleic acid including HIV DNA and mRNA into plasma. Production of viral proteins and packaging of viral genomes is a highly unlikely source given the hypermutated genome with at least one stop codon in gag-p24. Release of cellular nucleic acids into plasma may be an underappreciated cause of false virologic failure.

replication-competent virus by qVOA (37x10 6 CD4+ T cells), and total HIV-DNA in sorted CD4+ T cell subsets. In 9 LoViReTs with <0.1 infectious units per million (IUPM), total HIV-DNA was measured in rectal and/or lymph node biopsies (LN). Clinically matched individuals with HIV-DNA >50 HIV-DNA copies/10 6 PBMCs were recruited as controls. Results: LoViReT harbored significantly lower total HIV-DNA in CD4+ T cells before ART initiation compared to controls (1,051 and 5,995 HIV-DNA copies/10 6 CD4+ T cells respectively, p=0.002) despite comparable pre-ART viral load. These differences became higher after 5 years on ART (16 vs 5-folds decay respectively, p<0.001). 10/14 LoViReTs had undetectable replication-competent virus (IUPM<0.0185) >10 years after ART. Among them, we detected low levels of HIV-DNA in rectum in 6/8 subjects with a median of 57 HIV-DNA copies/10 6 CD45+ T cells [IQR:37-114]. In LN, only 3/8 subjects had detectable reservoir (263[36-2,112] HIV-DNA copies/10 6 CD45RA- T cells). Unexpected HIV reservoir distribution was observed in LoViReTs, being the short-live transitional memory (TTM) and effector memory (TEM) T cells the major contributors to the total reservoir (47% and 29% respectively). TCM presented limited contribution to the HIV reservoir (24%). Conclusion: LoViReT individuals have abnormally low HIV reservoirs before ART initiation. 71% of LoViReTs did not have replication-competent virus and harbored limited provirus in tissue sanctuaries after a median of 15 years on ART. A cause of this exceptional low reservoir could be the high contribution of the short-live TTM and TEM cells in the total HIV reservoir. This unique group of individuals are of great interest as trial participants in eradication studies. 375 A NEW LONG-READ NGS METHOD TO SEQUENCE HIV1 INSERTION SITES AND ASSOCIATED PROVIRUSES Maria Artesi 1 , Vincent Hahaut 2 , Basiel Cole 3 , Laurens Lambrechts 3 , Ambroise Marçais 4 , Olivier Hermine 4 , Philip Griebel 5 , Natasa Arsic 5 , Dominique Bron 6 , Carole Charlier 2 , Linos Vandekerckhove 3 , Michel Georges 2 , Anne Van den Broeke 6 , Keith Durkin 7 1 GIGA German Institute of Global and Area Studies, Liege, Belgium, 2 University of Liege, Liege, Belgium, 3 HIV Cure Research Center, Ghent University, Ghent, Belgium, 4 Hopital Necker Enfants Malades, Paris, France, 5 University of Saskatchewan, Saskatoon, Canada, 6 Institut Jules Bordet, Brussels, Belgium, 7 University of Liège, Liege, Belgium Background: The HIV-1 reservoir represents a major obstacle to HIV cure, making its exploration a priority, however, this task is complicated by its elusiveness, with only ~0.1% of CD4 T cells carrying integrated HIV-1 DNA. Substantial effort has been expended to determine the patterns of proviral integration in this latent reservoir and simultaneously identify the sequence of the associated HIV-1 proviruses. Recent approaches based on short-read high throughput sequencing allow the sequence of individual proviruses to be linked to the integration site, however, these methods rely on whole genome amplification of isolated HIV-1 genomes, with separate reactions to identify the integration site and sequence the provirus, limiting the number of proviruses one can reasonably interrogate. Methods: To exploit the potential of long reads we developed Pooled CRISPR Inverse PCR sequencing (PCIP-seq), a method that leverages selective cleavage of circularized DNA fragments carrying HIV-1 proviral DNA with a pool of CRISPR guide RNAs, followed by inverse long-range PCR and multiplexed sequencing on the Oxford Nanopore MinION platform. Results: We first tested PCIP-seq on 0.1 and 0.01% dilutions of the HIV-1 cell line U1 and demonstrated its utility to examine low proviral loads. We then applied PCIP-seq to CD4 T cells of two HIV-1 patients on long term cART, generating the sequence from hundreds of HIV-1 proviruses and linked this sequence to specific integration sites. We identified proviruses with single nucleotide variants and large deletions as well as intact proviruses. Among these, we found proviruses present in clonally expanded cells mapping to segmentally duplicated regions and satellite repeats of the centromeres of chr13, 14, 21 and 22. Both patients had four integration sites in intron 1 of STAT5B, all in the same transcriptional orientation as the host gene. In addition to HIV-1 we also successfully applied the technique to oncogenic retroviruses HTLV-1 and BLV. Conclusion: Using long reads, we can simultaneously identify the integration site and track clone abundance while also sequencing the HIV-1 provirus inserted at that position. Methods currently used are labor intensive, costly, and only examine a handful of patients. Using PCIP-seq it is feasible to sequence thousands of bases from hundreds of proviruses in a single experiment, opening

Poster Abstracts

374 ART-TREATED SUBJECTS WITH LOW VIRAL RESERVOIR SHOW UNUSUAL HIV LATENCY DISTRIBUTION Cristina Gálvez 1 , Victor Urrea 1 , Susana Benet 1 , Lucia Bailon 2 , Andrea Martinez 2 , Beatriz Mothe 2 , Judith Dalmau 1 , Lorna Leal 3 , Felipe Garcia 3 , Javier Martinez- Picado 1 , Maria Salgado 1 1 IrsiCaixa Institute for AIDS Research, Badalona, Spain, 2 Fundació Lluita Contra la Sida, Badalona, Spain, 3 Hospital Clinic of Barcelona, Barcelona, Spain Background: Small-size viral reservoirs are predominantly found in HIV-1 controllers and individuals treated during acute/early HIV-1 infection. However, other HIV+ subjects could naturally also harbor low viral reservoirs. We have established a cohort of "Low Viral Reservoir Treated" subjects" (LoViReT) to further explore the mechanisms associated with low reservoir levels. Methods: 42 HIV+ subjects on ART and <50 HIV-DNA copies/10 6 PBMCs constitute the LoViReT cohort; at least 66% of whom initiated ART during the chronic phase of HIV (>6 months since acquisition). In 12 LoViReTs, total HIV-DNA was longitudinally measured in cryopreserved CD4+ T cells by ddPCR, including a pre-ART time point. 14 LoViReTs underwent a leukapheresis to measure the

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