CROI 2020 Abstract eBook

Abstract eBook

Oral Abstracts

responses to antigens represent selective forces affecting the persistence of both defective and infectious proviruses. 74 DISTINCT CHROMOSOMAL INTEGRATION SITE CONFIGURATION IN HIV-1 ELITE CONTROLLERS Chenyang Jiang 1 , Ce Gao 1 , Xiaoming Sun 1 , Xiaodong Lian 1 , Stephane Hua 1 , Joshua Chevalier 2 , Kevin Einkauf 2 , Eric Serrao 3 , Alan N. Engelman 3 , Mary Carrington 4 , Bruce D. Walker 1 , Mathias Lichterfeld 2 , Xu G. Yu 1 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Brigham and Women's Hospital, Boston, MA, USA, 3 Dana–Farber Cancer Institute, Boston, MA, USA, 4 National Cancer Institute, Frederick, MD, USA Background: HIV-1 elite controllers (EC) represent a rare group (< 0.5%) of infected individuals who maintain undetectable viral loads in the absence of antiretroviral therapy (ART). However, the distinguishing features of proviral reservoir cells in these individuals are unclear. Methods: Matched integration site and proviral sequencing (MIP-Seq) was applied to PBMC from 11 EC to investigate chromosomal integration sites (IS) of intact HIV-1 proviruses. Chromatin accessibility and gene expression in autologous CD4 T cells were measured by ATAC-Seq and RNA-Seq. CD4 T cells from 12 EC and 11 HIV-1 negative individuals (HIVN) were infected with a HIV-1 construct, followed by chromosomal IS analysis. Results: In total, 92 IS of intact proviruses were identified in EC, of which 33 were at unique chromosomal locations. Remarkably, we noted that a significantly larger proportion of intact proviruses from EC were located in non-genic, centromeric satellite DNA, compared to 73 unique (100 in total) intact proviral sequences from long-term ART-treated individuals (unique IS: 21% vs. 0%, p=0.0002; all IS: 17% vs. 0%, p<0.0001). Moreover, in comparison to ART-treated patients, IS of intact proviruses from EC were atypically enriched in genes encoding for members of the Zinc Finger Protein family, particularly for KRAB-ZNF on chromosome 19, which contain constitutive heterochromatin (unique IS: 22% vs. 2%, p=0.0091; all IS: 40% vs. 1%, p<0.0001). In addition, we identified significantly increased chromosomal distances from IS of intact proviruses to the most proximal host gene transcriptional start sites (median: 29.3 kb vs. 9.4 kb, p=0.0002) and to accessible chromatin (median: 73.1 kb vs. 8.8 kb, p=0.0004) in CD4 T cells from EC, relative to ART-treated patients. Furthermore, >120,000 HIV-1 IS from in vitro infected CD4 T cells from EC and HIVN demonstrated that satellite DNA (0.04%-0.12%) and KRAB-ZNF genes (0.49%-0.85%) were infrequently targeted, irrespective of the study cohort. Conclusion: Integration sites of intact proviruses in EC show features of deep latency, likely as the result of selection mechanisms that preferentially eliminated proviruses integrated in chromosomal regions more permissive to viral transcription. This highly distinct chromosomal integration site configuration in EC represents a structural correlate of natural viral control that eradication strategies may have to induce in order to promote a long-term drug- free remission of HIV-1 infection. 75 INTACT PROVIRAL DNA LEVELS DECLINE IN PEOPLE WITH HIV ON ANTIRETROVIRAL THERAPY Rajesh T. Gandhi 1 , Joshua C. Cyktor 2 , Ronald Bosch 3 , Hanna Mar 3 , Gregory Laird 4 , Albine Martin 4 , Ann Collier 5 , Sharon Riddler 2 , Bernard J. Macatangay 2 , Charles Rinaldo 2 , Joseph J. Eron 6 , Robert Siliciano 7 , Deborah McMahon 2 , John W. Mellors 2 , for the ACTG A5321 team 1 Massachusetts General Hospital, Boston, MA, USA, 2 University of Pittsburgh, Pittsburgh, PA, USA, 3 Harvard T.H. Chan School of Public Health, Boston, MA, USA, 4 Accelevir Diagnostics, Baltimore, MD, USA, 5 University of Washington, Seattle, WA, USA, 6 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 7 Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: The intact proviral DNA assay (IPDA) is a new, more-specific ddPCR-based measure of the replication-competent HIV reservoir. Little is known, however, about whether intact proviral DNA levels decline over time on ART and whether the levels correlate with other measures of HIV persistence or with immune activation. Methods: Participants in ACTG A5321 with chronic HIV and well documented virologic suppression on ART had the following measurements performed on blood samples: intact proviral DNA, total proviral DNA (sum of defective, hypermutated and intact proviruses), total HIV DNA by qPCR targeting 3’ integrase, cell-associated HIV RNA (CA-RNA), plasma HIV RNA single copy assay (SCA), T cell activation, and inflammation (IL-6, IP-10, sCD14, sCD163, neopterin,

Oral Abstracts

73 ANTIGEN-DRIVEN CLONAL SELECTION SHAPES THE FATE OF HIV- INFECTED CD4+ T CELLS IN VIVO

Francesco R. Simonetti 1 , Hao Zhang 2 , Garshasb Soroosh 1 , Subul A. Beg 1 , Jiayi Duan 1 , Kyle Rhodehouse 1 , Christopher L. Nobles 3 , Jun Lai 1 , Rebecca Hoh 4 , Steven G. Deeks 4 , Frederic Bushman 3 , Janet Siliciano 1 , Robert Siliciano 1 1 Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2 Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA, 3 University of Pennsylvania, Philadelphia, PA, USA, 4 University of California San Francisco, San Francisco, CA, USA Background: Although proliferation of infected CD4+ T-cells is a major mechanism of HIV persistence, the causes of this phenomenon remain unclear. We hypothesized that recurrent antigenic exposure contributes, via clonal selection, to the expansion and maintenance of HIV-infected cells. Methods: We enrolled 10 HIV+/CMV+ donors on ART. PBMC were briefly stimulated with CMV lysates, GAG peptides or αCD3/28 antibodies. We sorted responding (CD40L+CD69+) and non-responding memory (CD40L-CD69- CD45ROhi) CD4+ T-cells. Single genome sequencing was used to identify identical proviruses. To study HIV-infected clones, we sorted cells in small pools at limiting dilutions and subjected to whole genome amplification. Proviruses in Ag-specific clones were analyzed by IPDA, integration site and full-length sequencing. We used TCR sequencing to study VDJ rearrangements in sorted cells and infected clones. A viral outgrowth assay (VOA) was used in one donor to identify clones carrying infectious proviruses. Results: PBMC stimulation yielded the expected frequencies of CMV- and GAG- specific CD4+ T-cells (mean 2.8% and 1.1%, respectively). Cells responding to non-specific CD3/28 stimulation (mean 33%) were used as a control. Proviruses in CMV-specific cells showed a higher proportion of identical sequences (0.73 vs 0.28, p<0.0001) and higher clonality (mean Gini 0.6 vs 0.2, p=0.0002) than the non-specific control. GAG-specific cells had detectable but less abundant identical sequences. Clonal proviruses were confirmed by integration site analysis. Some clones had integrants in genes previously identified in other individuals on ART, such as BACH2, STAT5B and MKL1. TCR sequencing confirmed a higher clonality of CMV-specific cells compared to GAG-specific and non- responding memory cells (mean clonality 0.2 vs 0.05 vs 0.03, respectively). Most clones carried defective proviruses (hypermutation, deletions, inversions). In one individual, the VOA from CMV-specific cells identified the same isolate in 4 wells that matched identical DNA sequences from CMV-specific cells collected 8 months previously, suggesting the persistence of a CD4 clone selected over time in response to CMV and that harbored an infectious provirus. Conclusion: We provide in vivo evidence that clonal expansion of HIV-infected cells is common for CMV- and GAG-specific CD4+ T-cells, demonstrating that

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CROI 2020