CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

283 FAST VALIDATION TO STUDY THE BASELINE AND INDUCIBLE HIV RESERVOIR Marilia R. Pinzone 1 , Xiaohe Liu 2 , Lidia Sambucetti 2 , Giuseppe Nunnari 3 , Una O’Doherty 1 1 Univ of Pennsylvania, Philadelphia, PA, USA, 2 SRI Intl, Menlo Park, USA, 3 Univ of Messina, Messina, Italy Background: “Shock and kill” strategies require highly sensitive methods to detect HIV reactivation upon stimulation with latency reversal agents (LRAs). In this study, we validated that Fiber-optic array scanning technology (FAST) can detect reactivation of latent HIV. Methods: Resting CD4 T cells from HIV-infected individuals or from PBMCs of uninfected donors were spinoculated with pNL4-3 virus or pLENG1-IRESGFP virus +/- an integrase inhibitor. Cells were cultured with IL-7 for 7 days and stimulated with several compounds, alone or in combination, such as prostratin (300 nM), bryostatin (1 µM), JQ1 (1 µM), or resveratrol (40 µM). After 24 hours, HIV gag or GFP expression was assessed by flow and FAST. Data are presented as mean fold increase of 3 separate experiments compared to the unstimulated control. Results: We validated FAST ability to detect LRA reversal by comparing it to flow using an in vitro model with a high frequency of infected cells. Stimulation with LRAs was associated with an increase in the number of GFP+ or gag+ cells (GPCs). Bryostatin was the most potent compound, inducing a consistent increase in the number of GPCs detected by FAST and flow (3.5- and 4-fold, respectively). Lower fold changes were detected by FAST and flow respectively when using prostratin (1.3-3.3 fold), JQ1 (4.1-1.9 fold), or resveratrol (1.8-3.3 fold). The association of bryostatin and JQ1 was synergistic in increasing the number of GPCs by FAST (5.7-fold increase) and flow (3.8-fold increase). Side by side comparisons of diluted in vitro infected cells as well as patient samples show FAST is more sensitive than flow. All patients had detectable levels of gag+ cells at baseline and after stimulation by FAST, but not by flow. Specifically, flow failed to robustly detect cells that express HIV proteins when the frequency of infected cells is below 1 in 10,000 due to inherent technical limitations. Conclusion: FAST detects HIV proteins expressed in resting CD4+ T cells at baseline and after stimulation providing a tool to assess HIV reactivation upon stimulation with LRAs. We find a correlation between FAST and flow at high inoculum, but flow loses sensitivity at low infection frequencies. FAST has benefits over flow cytometry, including the ability to capture events that can be missed by flow, such as syncytia of cells expressing HIV proteins. In conclusion, FAST represents a novel sensitive assay, which can help characterize the baseline and inducible reservoir of HIV-infected patients. 284 HOST TRANSCRIPTOMIC DETERMINANTS OF HIV PERSISTENCE IN VIVO Mohamed Abdel Mohsen 1 , Xutao Deng 1 , Sheila Keating 1 , AndrewWorlock 2 , Christopher D. Pilcher 3 , Michael P. Busch 1 , Steven G. Deeks 3 , Satish K. Pillai 1 1 Blood Systems Rsr Inst, San Francisco, CA, USA, 2 Hologic Corporation, Bedford, MA, USA, 3 Univ of California, San Francisco, San Francisco, CA, USA Background: HIV eradication will likely require identification of host factors that govern HIV latency in vivo. We performed global transcriptomic profiling of CD4+ T cells from HIV-infected, ART-suppressed individuals to identify host determinants of HIV persistence. Methods: We measured levels of cell-associated (CA) HIV RNA, and total pol and 2-LTR circle HIV DNA in CD4+ T cells from 69 HIV-infected individuals on suppressive ART for 1-2 years; 23 subjects initiated ART <6 months post-infection (“Early Tx”), and 46 subjects initiated >1 year post-infection (“Chronic Tx”). Residual plasma HIV RNA levels were measured using an ultrasensitive, real-time transcription-mediated amplification-based assay, and circulating anti-HIV-1 antibodies were characterized using a limiting antigen assay. We performed flow cytometry and RNA-seq to characterize the immunophenotype and global transcriptome of isolated CD4+ T cells, respectively. False discovery rate (FDR) was used to correct for multiple comparisons in statistical analyses. Results: Low-level viremia was detected in 19 out of 23 Early Tx (0.5±0.37, mean log copy number ±SD) and 34 out of 46 Chronic Tx individuals (0.46±0.3); detectable frequencies and levels were similar between the two groups. Residual viremia was correlated with expression of the host transcriptional mediator ZBTB1 (Zinc finger and BTB domain containing 1) (FDR<0.005). Levels of CA HIV RNA were correlated with multiple factors belonging to the AP-1 (activator protein-1) transcription factor complex (FDR <0.05). The quantity and avidity of anti-HIV antibodies were higher in the Chronic Tx group as compared to Early Tx (p<0.0001). 71 genes were differentially expressed between the Early Tx and Chronic Tx groups (FDR <0.05), including several genes associated with mitochondrial dysfunction, oxidative phosphorylation, and the histone h3, IL12, T-cell receptor, p38 mitogen-activated protein kinase, and PI3K-Akt complex families. Conclusion: Residual viremia was readily detectable in the majority of ART-suppressed individuals. The level of viremia was similar in those treated during early versus chronic infection, despite lower levels of immune activation and CA HIV RNA and DNA in those treated early. Several pathways were differentially expressed in the Early Tx group and may contribute to the smaller reservoir size observed in the setting of early ART. Our transcriptomic signatures reveal host factors and pathways that may be targeted to develop curative strategies for HIV infection.

Poster and Themed Discussion Abstracts

285 LOW INDUCIBILITY OF THE HIV RESERVOIR IN EARLY ART-TREATED THAI CHILDREN Marta Massanella 1 , Jintanat Ananworanich 2 , Louise Leyre 3 , Thidarat Jupimai 4 , Mark de Souza 5 , Piyarat Suntarattiwong 6 , Pope Kosalarksa 7 , Thanyawee Puthanakit 8 , Nicolas Chomont , for the HIVNAT209 and HIVNAT194 Study Groups 1 Cntr de Rsr du Cntr Hosp de l’Univ de Montréal, Montréal, Canada, 2 Henry M. Jackson Fndn for the Advancement of Military Med, Bethesda, MD, USA, 3 HIV–NAT Rsr Collab, Thai Red Cross AIDS Rsr Cntr, Bangkok, Thailand, 4 SEARCH, Thai Red Cross AIDS Rsr Cntr, Bangkok, Thailand, 5 Queen Sirikit Natl Inst of Child Hlth, Bangkok, Thailand, 6 Khon Kaen Univ, Khon Kaen, Thailand, 7 Chulalongkorn Univ, Bangkok, Thailand Background: Early antiretroviral therapy (ART) limits the size of the HIV reservoir in adults, however pediatric data are limited. We evaluated the impact of early ART initiation on the size of the HIV reservoir in vertically infected infants.

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