CROI 2016 Abstract eBook

Abstract Listing

Poster Abstracts

358LB T-Cell Homeostasis and CD8 Responses Predict Viral Control Post SB-728-T Treatment Joumana Zeidan 1 ; Gary K. Lee 2 ; Clarisse Benne 1 ; Slim Fourati 1 ; Shelley K.Wang 2 ; Jacob Lalezari 3 ; Gary Blick 4 ; Geoffrey M. Nichol 2 ; Dale K. Ando 2 ; Rafick Sekaly 1 1 Case Western Reserve Univ, Cleveland, OH, USA; 2 Sangamo BioSciences, Richmond, CA, USA; 3 Quest Clinical Rsr, San Francisco, CA, USA; 4 CIRCLE CARE Cntr, LLC, Norwalk, CT, USA Background: Infusion of ZFN driven CCR5 modified T-cells (SB-728-T) in HIV infected subjects was previously shown to durably improve CD4 counts. Long-term CD4 reconstitution and decay of the HIV reservoir were previously correlated with a novel memory stem cell (TSCM) CD4 subset that expands post-infusion and is enriched in CCR5-modified cells. This current study aims at identifying correlates and mechanisms that lead to control of viremia post treatment interruption (TI). Methods: 18 ART treated subjects with CD4 counts above 500 were preconditioned with 0.1-2.0 g/m 2 of Cytoxan prior to infusion of SB-728-T. Subjects initiated TI at 6 weeks post infusion. T cell count and viral load (VL) were monitored regularly for 12 months following treatment. Immunologic and viral analyses (immuno-phenotyping, reservoir analyses, anti-HIVgag effector functions, etc) were performed at selected time points. Results: Of the 9 subjects pre-conditioned with Cytoxan doses of 1.0 and 1.5 g/m 2 , 6 subjects demonstrated durable control of viremia (VL<10,000) in extended TI (duration= 14-26 months), with 2 subjects showing consistent ongoing VL measurements <1000 (duration= 17 & 20 months). Using a univariate linear regression model, greater levels of CCR5 modified cells before TI (p=0.03) and frequencies of CD4 TSCM during TI (p=0.01) correlated with lower VL, suggesting that greater levels of the HIV resistant T-cell compartment could be critical in conferring post-treatment control possibly by restoring immune homeostasis and providing help to HIV specific CD8 T cells. Multivariate analyses were used to determine parameters that further predict viremia control during TI. Results indicate that higher CD4 TSCM levels, along with greater polyfunctional anti-HIV gag CD8 response during TI (p=0.04) were associated with reduced viral load. Further, HIV reservoir size prior to TI showed a significant interaction with CD8 response in this model (p=0.03), suggesting that greater HIV reservoir were associated to HIV specific CD8 responses that failed to control virus upon TI. Conclusions: Control of viremia during treatment interruption was demonstrated in 6 subjects treated with CCR5 modified T-cells. In a multivariate model, data from immunologic and reservoir analyses suggest that the best predictors for post treatment viral control are restored T cell homeostasis as suggested from greater levels of SB-728-T engraftment, polyfunctional antiviral CD8 responses during TI and lower HIV reservoir levels prior to TI. 359 Integration Analysis of Latently Infected Cell Lines: Evidence of Ongoing Replication Jori Symons 1 ; Abha Chopra 2 ; Eva Malantinkova 3 ;Ward Spiegelaerde 3 ; Shay Leary 2 ; Don Cooper 2 ; LinosVandekerckhove 3 ; Simon Mallal 2 ; Sharon R. Lewin 1 ; Paul U. Cameron 1 1 Doherty Inst for Infection and Immunity, Univ of Melbourne, Melbourne, Australia; 2 Inst for Immunology & Infectious Diseases, Murdoch Univ, Murdoch, Australia; 3 Ghent Univ and Ghent Univ Hosp, Ghent, Belgium Background: HIV cure is limited by persistence of long lived latently infected CD4 + T cells. Latently infected cell lines are widely used in vitro to study HIV latency. We identified and tested the stability of HIV integration sites in latently infected cell lines, obtained from NIH AIDS reagent program, using a newly developed high throughput method. Methods: Method: To determine assay sensitivity/efficiency, high molecular genomic DNA of seven latent HIV cell lines were isolated and DNA equivalent of 20 cells each were mixed with genomic DNA from a million HIV negative PBMCs. Additionally, the latently infected cell lines ACH-2, U1 and J1.1 that contain 1 or 2 integrated copies of replication competent HIV and J-Lat 8.4, 9.2, 10.6 and 15.4 cell lines that contain a single replication deficient HIV were passaged 8 times. Genomic DNA isolated after passage 0, 2, 4, 6 and 8 was enzymatically cut to random sized fragments. The fragments were end-repaired, a-tailed and a linker was ligated to the fragments which enables nested amplification. The fragments were subjected to LTR based nested PCR with barcoded nested primers and prepared for Miseq sequencing. Chromosomal alignment was determined using the Blat- UCSC Genome Browser (GRCH38/hg38). Results: The procedure was optimized for robotic processing to increase sample throughput and decrease risk of contamination. The efficiency was 35% and detected 1 HIV integration site in 50,000 uninfected PBMCs. Latently infected cell lines infected with a deficient virus demonstrated a single integration site. In contrast, cell lines harbouring a replication competent virus demonstrated multiple distinct HIV integration sites per 150,000 cells (74 in ACH2, 42 in U1 and 93 in J1.1). J1.1, which is reported to have a single integrated copy per cell, demonstrated two major integration sites in equal frequency. Moreover, ACH-2 cells, when passaged, demonstrated a 2-fold increase in unique HIV integration sites found across the human genome. Conclusions: Conclusion: Cell lines latently infected with replication competent HIV demonstrated multiple unique HIV integration sites indicating these cell lines are not clonal. Furthermore, the increase and change in sites of HIV integration observed in ACH-2 cells over time is suggestive of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and as standards for quantification of HIV integration. 360 HIV Integration Sites in Cellular Models of Viral Persistence Sara Sunshine 1 ; Rory Kirchner 2 ; Sami S. Amr 3 ; Michelle Kim 4 ; Alberto Bosque 5 ; Robert Siliciano 6 ;Vicente Planelles 5 ; Oliver Hofmann 2 ; Shannan Ho Sui 2 ; Jonathan Z. Li 1 1 Brigham and Women’s Hosp, Harvard Med Sch, Boston, MA, USA; 2 Harvard Sch of PH, Boston, MA, USA; 3 Partners HlthCare Personalized Med, Cambridge, MA, USA; 4 Johns Hopkins Univ Sch of Med, Baltimore, MD, USA; 5 Univ of Utah, Salt Lake City, UT, USA; 6 Howard Hughes Med Inst, Baltimore, MD, USA Background: The location of HIV integration sites is thought to play a critical role in the clonal expansion of infected cells and HIV persistence. Cell culture models are often used to study and assess HIV latency and latency-reversing agents, but it is unclear how these models reflect HIV integration in vivo. We have developed a high-throughput sequence- capture assay to evaluate the integration site characteristics of 3 cellular models. Methods: Integration site analysis was performed in three cellular models of HIV persistence: ACH-2 cells, Bcl-2 transduced primary CD4+ cells, and cells from a cultured T CM model. A clonal population of ACH-2 cells was also included after limiting dilution and expansion with ART. Sequence enrichment was performed with a diverse biotinylated probe library composed of 52 consensus probe sequences designed using the LANL database. Libraries were sequenced on the MiSeq platform (Illumina) with 300 bp paired-end reads. Sites were annotated with UCSC canonical gene annotations and gene ontology analysis was performed using the Gene SeT Analysis Toolkit. Genes identified from the cellular models were also compared to highly represented genes from two previously published patient datasets. Results: While ACH-2 cells are historically thought to have a single site of integration, we identified two main integration sites. In addition, we found that 47% of reads were outside of the two major integration sites. Integration site analysis of the clonal ACH-2 population showed near elimination of single integration sites (ACH-2 bulk culture vs. ACH-2 clone: 39.4% vs. 0.2%, Fisher’s P <0.01), suggesting that this assay has an extremely low false-positive rate and confirming the diversity of integration sites within the ACH-2 population. Across the 3 cellular models, there were significant differences in integration site characteristics, including the proportion of clonally-expanded sites, sites within genic regions, exons, and the orientation of the integrated HIV relative to the host gene. Gene ontology analysis of highly represented genes from the patient samples found little overlap with HIV-containing genes from the cell lines. Conclusions: Integration site differences exist amongst the commonly used cellular models of HIV persistence and in comparison to integration sites found in patient samples. These results may contribute to differences seen in the response to latency-reversing agents amongst cellular models and patient-derived cells. 361 Clonal Integration Site Frequency and Replication-Competent Virus in Patients on ART Maja Kiselinova 1 ;Ward Spiegelaerde 1 ; Maria J. Buzon 2 ; Sherry McLaughlin 3 ; Mathias Lichterfeld 2 ; Lisa Frenkel 3 ; LinosVandekerckhove 1 1 Ghent Univ and Ghent Univ Hosp, Ghent, Belgium; 2 Ragon Inst of MGH, MIT, and Harvard, Cambridge, MA, USA; 3 Seattle Children’s Rsr Inst, Seattle, WA, USA Background: The latent HIV-1 reservoir remains one of the major obstacles to cure HIV. To monitor HIV cure strategies, a validated biomarker is needed that can evaluate the reservoir over time in vivo . Consequently, numerous assays are now being investigated to estimate the size of the replication competent provirus. Methods: A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 ART treated patients was sampled in which plasma viral load (<50 cp/ml) was suppressed for median of 7 years (5-11). Total, integrated HIV-1 DNA and unspliced (us-) HIV-1 RNA were quantified in peripheral blood. A quantitative

Poster Abstracts

137

CROI 2016