High-throughput Deep Sequencing Assay to Reliably Measure the HIV Reservoir during Antiretroviral Therapy

University Of California, San Diego, La Jolla CA

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Abstract

? DESCRIPTION (provided by applicant): This study is designed to meet the goals of the RFA Innovative assays to quantify the latent HIV reservoir that can facilitate proof-of-concept studies for curing HIV infection. To meet this challenge we propose to develop and validate a combined next generation sequencing (Pacific Biosciences single-molecule, real time (SMRT) sequencing) and bioinformatics platform to accurately measure replication-competent provirus (i.e. Proviral Phenotypic Predictor; P3). An over-arching objective would be for the assay to have a higher throughput, faster turnaround, lower cost, and greater reproducibility than the current standard quantitative viral outgrowth assay (QVOA). We will develop and validate the proposed P3 platform based on international standard procedures for diagnostic molecular methods (i.e. CAP and OIE) using latently infected cells from HIV-infected individuals on optimized antiretroviral therapy for at least 2 years. These participants will be well-characterize to estimated duration of infection at the start of their therapy, and will represent high, medium and low levels of HIV DNA levels. Such characterizations will be necessary to adequately address the dynamic range of the proposed P3 platform. Once P3 methods have been optimized, we will rigorously compare P3 and QVOA methods in their ability to characterize the replication competent proviral reservoir and by costs and turnaround time for results.