Abstracts of the 9th IAS Conference on HIV Science (IAS 2017) Journal of the International AIDS Society 2017, 20 (Suppl 5) http://www.jiasociety.org/index.php/jias/article/view/22253 | http://dx.doi.org/10.7448/IAS.20.6.22253
ORAL ABSTRACTS MOAA0101 Assessing individual viral reactivations of the latent reservoir using a novel barcoded virus C Fennessey1; M Pinkevych2; T Immonen1; C Camus1; G Del Prete1; J Estes1; J Lifson1; M Davenport2 and B Keele1 1 Leidos Biomedical Research Inc., Frederick National Laboratory, AIDS and Cancer Virus Program, Frederick, USA. 2Kirby Institute for Infection and Immunity, University of New South Wales, Sydney, Australia Presenting author email: [email protected]
Background: An overwhelming obstacle to HIV cure is the presence of long-lived viral reservoirs that can reignite viral infection after removal of combination antiretroviral therapy (cART). Yet, our knowledge of the mechanisms of reservoir maintenance and persistence remains incompletely understood. Therefore, in this study, we generated a novel virus model for evaluating individual reactivation events following cART interruption to better understand key aspects of reservoir biology in vivo. Methods: A genetically tagged virus (SIVmac239M) was generated with a 34-base molecular barcode stably inserted between vpx and vpr of SIVmac239. Rhesus macaques were infected intravenously and cART was administered, beginning either on day 6 for 82 days (n = 4) (study 1) or on day 4 for 305, 374 or 482 days (n = 6) (study 2). Nextgeneration sequencing was used to evaluate the number of genetic variants in the stock and in plasma before and after treatment. Results: In the viral stock, 9336 individual barcodes, or clonotypes, were identified. During acute infection, an average of 1247 barcodes were identified in each of the 10 animals. Plasma viraemia was reduced to 15 vRNA copies/ml during treatment, and virus rapidly rebounded following treatment interruption. Between 87 and 136 distinct clonotypes were detected in plasma at peak rebound viraemia in animals from study 1 and between 4 and 7 clonotypes in animals from study 2. Because the growth rate of each clonotype is equivalent once the virus reaches systemic infection, the measured relative proportions of each clonotype at rebound reflect the time between when each clonotype achieved systemic infection. The viral growth rate and the relative abundance of each clonotype in plasma during acute recrudescence were used to estimate a reactivation rate of 22.7 and 0.54 events per day in studies 1 and 2, respectively. Conclusions: We conclude that identifying rebounding clonotypes may be used as a direct measurement of the latent reservoir that can successfully contribute to rebound viraemia. Furthermore, the results confirm that the size of the post-cART recrudescence-competent viral reservoir is influenced by the timing of cART initiation and duration of treatment, enabling manipulation of these parameters to establish reservoirs of desired size for specific experimental purposes.
MOAA0102 Accumulation and persistence of deleted HIV proviruses following prolonged ART E Anderson1; S Hill1; J Bell2; F Simonetti1; C Rehm3; S Jones4; R Gorelick2; J Coffin5 and F Maldarelli1 1 National Cancer Institute, HIV Dynamics and Replication Program, Frederick, USA. 2Leidos, Biomedical Research Inc, Frederick, USA. 3 National Institutes of Allergy and Infectious Diseases, Laboratory of Immunoregulation, Bethesda, USA. 4Leidos, Biomedical Research Inc, Clinical Monitoring Research Program, Frederick, USA.
Department of Molecular Biology and Microbiology, Tufts University, Boston, USA Presenting author email: [email protected]
Background: HIV persistence during antiretroviral therapy (ART) is a substantial obstacle to HIV cure. HIV-infected cells can undergo clonal expansion, and specific clones may be highly expanded. We previously reported one provirus integrated in the HORMAD2 gene that accounted for 20–40% of all proviruses in one patient. Mechanisms by which clones emerge and persist over time are uncertain. To investigate the dynamics of HIV clonal expansion, we developed multiplexed droplet digital approaches (ddPCR) to quantify HIV proviruses, including specific integrants, prior to and following prolonged ART. Methods: HIV-infected ART-naive individuals (N = 11) underwent ART and were followed for a median of 13.7 years (range 4.3–16.4). Cell-associated DNA (CA-DNA) from peripheral blood lymphocytes pre-ART, during first- and second-phase viral decay and after prolonged ART was quantified using multiplexed ddPCR assays targeting HIV gag, LTR and tat/rev regions, as well as a host gene (CCR5). We designed a specific ddPCR primer set overlapping the host–HIV junction to quantify the HIV integrant in HORMAD2. Results: All patients had successful suppression of HIV RNA on ART to