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.
This proposal is specifically designed to develop and validate a high-throughput next generation sequencing and bioinformatics platform (P3='Proviral Phenotypic Predictor') to fully characterize the latent HIV reservoir through quantification and sequencing of Full-length HIV provirus. We will determine the accuracy and exact error rate for our proposed platform, and we will compare this new assay to the standard quantitative viral outgrowth assay (QVOA) to delineate what amount of intact and replication competent HIV genomes is not inducible (and therefore not recognized) by QVOA. Being able to characterize each part of the HIV DNA population, and especially determine what proportion of intact and replication competent HIV DNA is not inducible by standard QVOA, will allow us to better evaluate the impact of any intervention aimed at reducing the HIV latent reservoir. 1
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