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

 描述（由申请方提供）：本研究旨在满足RFA“定量潜伏HIV库的创新检测”的目标，该目标可以“促进治疗HIV感染的概念验证研究”。“为了应对这一挑战，我们建议开发和验证组合的下一代测序（太平洋生物科学公司单分子，真实的时间（SMRT）测序）和生物信息学平台，以准确测量复制能力的前病毒（即前病毒表型预测; P3）。一个过度的目标将是该测定具有比当前标准的定量病毒生长测定（QVOA）“更高的通量、更快的周转、更低的成本和更大的再现性”。我们将根据诊断分子方法的国际标准程序（即CAP和OIE），使用来自接受优化抗逆转录病毒治疗至少2年的HIV感染者的潜伏感染细胞，开发和验证拟议的P3平台。这些参与者将在治疗开始时充分表征估计的感染持续时间，并将代表高、中、低水平的HIV DNA水平。这种表征对于充分解决所提议的P3平台的动态范围是必要的。一旦P3方法得到优化，我们将严格比较P3和QVOA方法在表征可复制前病毒库的能力以及结果的成本和周转时间方面。

 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.