CROI 2019 Abstract eBook

Abstract eBook

Oral Abstracts

We performed flow cytometric analysis of T cell activation and assessed the gene expression profile and SIV-specific T-cell responses following AZD5582 treatment. Results: Treatment with AZD5582 resulted in efficient activation of ncNF-κB in absence of generalized T-cell activation in blood and LN. A persistent increase in PVL on ART was observed in 5/12 (42%) AZD5582-treated RM while PVL remained undetectable in 9 control animals. The episodes of viremia induced by AZD5582 started as soon as 48h after the first dose. Viremia >60 copies/ml was measured in 15/28 samples (53%) in a period of 10 weeks with levels reaching 1390 SIV-RNA copies/ml. The levels of cell-associated SIV-RNA in resting CD4+ T-cells isolated from LN were significantly higher in 10-dose AZD5582-treated animals vs. controls (p= 0.0157) and tended to also be higher in the spleen, but not blood or BM. The levels of SIV-DNA quantified in the same compartments were not significantly different between AZD5582-treated and control groups. Principal component analyses revealed a distinct impact of AZD5582 on the transcriptome of CD4+ T cells isolated from blood and LN pre- and post- treatment. SIV-specific T cell responses measured in blood and LN by ELISPOT were not negatively impacted by treatment with AZD5582. Conclusion: Activating the ncNF-κB pathway in vivo with the SMACm AZD5582 resulted in high level and persistent induction of SIV-RNA expression in ART-suppressed RM in absence of generalized T-cell activation, indicative of latency reversal. Further studies will combine this promising LRA with immune clearance strategies to reduce viral reservoirs. 23 NONSUPPRESSIBLE VIREMIA ON ART FROM LARGE CELL CLONES CARRYING INTACT PROVIRUSES Elias K. Halvas 1 , Kevin Joseph 1 , Leah D. Brandt 1 , Johannes C. Botha 2 , Michele Sobolewski 1 , Jana L. Jacobs 1 , Brandon F. Keele 3 , Mary F. Kearney 4 , John M. Coffin 5 , Jason W. Rausch 4 , Shuang Guo 6 , Xiaolin Wu 6 , Stephen H. Hughes 4 , John W. Mellors 1 1 University of Pittsburgh, Pittsburgh, PA, USA, 2 Stellenbosch University, Cape Town, South Africa, 3 AIDS and Cancer Virus Program, Frederick, MD, USA, 4 National Cancer Institute, Frederick, MD, USA, 5 Tufts University, Boston, MA, USA, 6 Leidos Biomedical Research, Inc, Frederick, MD, USA Background: Clinically detectable viremia on ART is generally attributed to virus replication from incomplete adherence and/or drug resistance. One case of infectious viremia from a large cell clone with an intergenic intact provirus has been reported in an individual with metastatic cancer (Simonetti, PNAS 2016). We studied individuals referred for clinically detectable viremia despite receiving potent ART, adherence counseling, and in some cases, regimen switches or intensification. Methods: Peripheral blood mononuclear cells (PBMCs) and plasma were collected at two or more time points from donors with plasma HIV RNA >20 copies/ml occurring for >6 months on combination ART. Single-genome sequencing was performed on plasma HIV RNA, cell-associated HIV DNA (CAD), and p24+ culture supernatants from quantitative viral outgrowth assays (qVOA). The clonal cellular origin of viremia was assessed by phylogenetics and integration site analysis (ISA), and confirmed by sequencing the integrated provirus and the flanking host sequences. Results: Across the 10 individuals referred, median plasma HIV-1 RNA was 97.5 cps/mL (range 40 to 356 cps/mL) after a median of 10 years on ART. One donor (A-04) had phylogenetic evidence of virus evolution and accumulation of resistance mutations and was not analyzed further. Each of the other 9 donors had multiple identical single-genome HIV RNA sequences in plasma that did not change over time and lacked resistance to the current ART regimen. In 6 of 9 donors, HIV sequences from plasma matched proviral sequences in PBMC. Plasma HIV RNA and proviral sequences were identical to HIV RNA in p24+ qVOA wells for 4 donors (C02, C03, R09, T13). The integration sites for the intact proviruses producing viremia were mapped to the human genome for 3 donors (4th pending). Integrations were in introns of the MATR3, ZNF268, and ABCA11P genes for C02, C03, and R09, respectively. The provirus in MATR3 and ZNF268 were in the opposite orientation to the gene, whereas the ABCA11P integrant was in the same orientation. The intact provirus comprised 4.2-15.4% of all proviruses in PBMC with amplifiable pro/pol sequences. Conclusion: Large cell clones carrying intact proviruses can produce clinically relevant levels of viremia and should be considered in managing patients. The mechanisms involved in clonal expansion and persistence of cells with intact proviruses that produce viremia need to be understood to effectively target the HIV reservoir.

Oral Abstracts

24 EX VIVO AND IN VIVO EDITING OF THE SIV GENOME IN NONHUMAN PRIMATES BY CRISPR-CAS9 Tricia H. Burdo 1 , Pietro Mancuso 1 , Rafal Kaminski 1 , Jennifer Gordon 1 , Binhua Ling 2 , Andrew MacLean 2 , Kamel Khalili 1 1 Temple University, Philadelphia, PA, USA, 2 Tulane National Primate Research Center, Covington, LA, USA Background: Antiretroviral therapy (ART) has increased survival, but is a non-curative approach as replication competent proviral DNA, with high risk for reactivation upon ART cessation, remains. As such, HIV is now a chronic disease with a broad range of co-morbidities and drug toxicity. Curative strategies to eradicate the infected cells or viral genome without further treatment are vital. Here, we develop and test the ability of the CRISPR-Cas9 gene editing method for elimination of the SIV viral genome in rhesus macaques. Methods: We employed AAV-9 as a vector to deliver CRISPR-Cas9 designed to target sequences spanning the LTR and Gag genes and permanently inactivating proviral DNA by excising intervening DNA fragments. Adult Chinese rhesus macaques (n=8) were i.v. infected with SIVmac239. At 8 weeks post infection, animals were treated daily with a drug regimen of tenofovir, emtricitabine and dolutegravir (5.1/50/2.5mg/kg daily by s.q.). Ex vivo gene editing was performed in PBMCs by AAV9-CRISPR-Cas9 transduction, PCR amplification and Sanger sequencing of the amplicons to assess the potency and precision of viral DNA elimination. In a proof of concept in vivo study, 4 animals, 3 were given an i.v. infusion of AAV-9-CRISPR-Cas9 (10^13GC/kg), and after three weeks, animals were necropsied, blood and tissues were harvested virological and gene excision evaluations. Results: In all SIV-infected animals, ex vivo excision of viral DNA was confirmed by the detection of distinct DNA fragments of 464bp and 358bp resulting from the removal of intervening DNA sequences between 5’LTR to Gag and 3’LTR to Gag, respectively. Results from Sanger sequencing confirmed the breakpoint of the viral DNA. Delivery was confirmed by the presence of Cas9 and expression of both gRNAs. In vivo, both 5’LTR to Gag and 3’LTR to Gag excision were confirmed in blood of animals that received AAV-9-CRISPR-Cas9 infusion. In contrast to the control animal, which displayed rapid viral outgrowth, no outgrowth was detected in PBMC/CEM co-cultures after 30 days from animals with AAV-9- CRISPR-Cas9. Conclusion: We demonstrated, for the first time, high specificity and efficacy of the CRISPR technology for targeting SIV proviral LTR and Gag regions, which led to both ex vivo and in vivo editing of SIV DNA. These observations support the potential use of CRISPR/Cas9 technology as a curative strategy that warrants further investigation. 25 DELAYED VIRAL REBOUND DURING ATI AFTER INFUSION OF CCR5 ZFN- TREATED CD4 T CELLS Pablo Tebas 1 , Julie Jadlowsky 1 , Pamela Shaw 1 , Gary Lee 2 , Dale Ando 2 , Sukyung Kim 1 , SoeYu Naing 1 , Simon Lacey 1 , Bruce L. Levine 1 , Don L. Siegel 1 , Carl H. June 1 , James L. Riley 1 1 University of Pennsylvania, Philadelphia, PA, USA, 2 Sangamo Therapeutics, Inc., Richmond, CA, USA Background: Autologous CD4 T cells modified using CCR5 specific Zinc Finger Nucleases (ZFN) have a survival advantage in the presence of HIV, but the levels of modification are insufficient to control viremia (NCT00842634). The main

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CROI 2019