CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

specific adaptations with limited evidence for reversion of adaptations in non-selective environments suggestive of extensive compensatory networks. The antigen-specific T cell responses in the child overall suggested the immune response to the heavily pre-adapted HIV strains may focus on sub-dominant T cell epitopes as evidenced by de novo adaptation following transmission. Interestingly, there was evidence of cross-reactive T cells to the adapted and non-adapted form of an epitope at the TCR family level for the mother/child pair, but this did not extend to the α/β CDR3. Unsupervised clustering of scRNAseq data separated cells stimulated by the adapted and non-adapted forms, with common differentially expressed genes upregulated in both the mother and child. Conclusion: Such targets will be important in the development of a therapeutic vaccine for individuals that have an established reservoir of adapted virus. 185 DAILY IMMUNOLOGICAL/VIROLOGICAL VARIATIONS IN AVIREMIC ART- TREATED HIV PARTICIPANTS Debashree Chatterjee 1 , Tomas Raul Wiche Salinas 1 , Yuwei Zhang 1 , Delphine Planas 1 , Amelie Cattin 1 , Augustine Fert 1 , Etiene Moreira Gabriel 1 , Laurence Raymond Marchand 1 , Josee Girouard 2 , Nicolas Cermakian 3 , Daniel E. Kaufmann 1 , Jean-Pierre Routy 4 , Petronela Ancuta 5 1 Université de Montréal, Montreal, QC, Canada, 2 McGill University Health Centre Research Institute, Montreal, QC, Canada, 3 McGill University, Montreal, QC, Canada, 4 McGill University Health Centre, Glen site, Montreal, QC, Canada, 5 Centre de Recherche du CHUM, Montreal, QC, Canada Background: Biological functions fluctuate in a circadian manner to align with environmental changes. In healthy uninfected individuals, variations in T-cell trafficking are documented in the blood, with nadir CD4 counts in the morning. Daily variations are also observed for plasma cortisol and melatonin, two regulators of immune functions. HIV infection is associated with profound alterations in CD4 T-cell homeostasis and chronic immune activation. HIV transcription is regulated by BMAL1, a circadian clock master regulator. However, daily variations in immunological/virological parameters during ART- treated HIV infection remain unknown. Methods: Eleven ART-treated people living with HIV (PLWH; median CD4 counts: 606 cells/ml; age: 57 years; time since infection: 242 months; aviremia under ART: 216 months) were hospitalized at the CRCHUM Phase I Clinic a Friday afternoon for 40 hours. Starting the next morning, blood was collected/ processed every 4 hours for 24 hours before food intake. Polychromatic flow cytometry allowed cell counting/phenotypic analysis on fresh blood. Plasma levels of cortisol/melatonin and markers of mucosal barrier impairment (FABP2, LBP) were measured by ELISA. PBMC were frozen. HIV DNA/RNA were quantified by PCR on sorted CD4+ T-cells. Results: The memory/naive/regulatory T-cell counts showed daily variations, with maximal counts observed 20:00-4:00 (nadir 12:00). The expression of the HIV co-receptors CCR5/CXCR4, gut-homing molecules CCR6/integrin ß7, and the immune checkpoint PD-1 on memory T-cells showed similar maximal expression 20:00-4:00. Pro-inflammatory non-classical monocyte counts were similarly high 8:00-00:00 but dropped significantly at 4:00. Plasma FABP2 levels peaked at 4:00, while LBP levels significantly dropped at 4:00. Daily variations in plasma cortisol (peak 4:00-8:00) and melatonin (peak 4:00) levels were observed. HIV-DNA reservoirs were stable. HIV-RNA levels in CD4 T-cells collected at night were higher compared to morning. Conclusion: Daily variations in the blood T-cell/myeloid compartments, mucosal permeability markers, HIV transcription, and melatonin/cortisol levels, were observed in a cohort of aviremic ART-treated PLWH. These findings provide a rationale for studying the role of the circadian clock machinery in regulating residual HIV transcription under ART.

Poster Abstracts

186 Withdrawn 187 HIGH-THROUGHPUT SINGLE-MOLECULE SEQUENCING TO CHARACTERIZE AB-RESISTANT HIV/SHIV Sung Hee Ko 1 , Divya Kilam 1 , Mangaiarkarasi Asokan 1 , Dylan Westfall 2 , Amy Ransier 1 , Sam Darko 1 , Daniel Douek 1 , Richard A. Koup 1 , John R. Mascola 1 , James Mullins 2 , Eli A. Boritz 1 1 NIH, Bethesda, MD, USA, 2 University of Washington, Seattle, WA, USA Background: Although HIV-specific broadly-neutralizing antibodies (bNAbs) can suppress viremia in ART-naïve people, clinical use of bNAbs is limited by neutralization-resistant viruses that may be too rare for detection before bNAb infusion. Novel assays to track the dynamics of rare viruses after bNAb infusion may inform future bNAb treatment approaches, and may also allow a better understanding of HIV evolution in response to humoral immune pressure. Methods: We optimized high-throughput, single-copy HIV env sequencing methods to study samples taken in bNAb infusion trials. Virion RNAs were reverse-transcribed with or without the addition of 8-nucleotide unique molecule identifiers (UMIs), followed by PCR. Pacific BioSciences single-molecule, real-time (SMRT) technology was used to obtain full-length env sequences. Sequence data were analyzed using standard and custom software tools. Errors arising in the sequencing process were quantified using data obtained from HIV molecular clones and HIV-infected participant plasma virus samples. Results: Initial studies demonstrated concordance of non-UMI-based SMRT sequence data with Sanger sequence data obtained in parallel from three HIV-infected participants. A non-UMI-based approach was then used to study samples from SHIV-infected macaques treated with bNAb VRC07-523LS. We observed pronounced changes in env sequences after VRC07-523LS infusion, with predominance of entirely new env clades and a relative loss of species clustering with pre-infusion virus. Selection of amino acid variants at several positions associated with resistance to CD4-binding-site antibodies was observed. Using plasmid HIV clones, we found that most of the error in the sequencing process was generated during the PCR and sequencing steps. We found that the use of UMIs reduced errors to a rate consistent with error rate of the RNA reverse transcription step alone. The number of unique sequences obtained after UMI-based analysis was comparable to the input template number, and reconstruction experiments showed that the use of UMIs substantially eliminated sequencing errors.

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