Simplified Assays of Latent But Inducible HIV-1 Reservoirs

描述（由申请人提供）：目前正在积极寻求通过接种疫苗或暴露前预防来预防获得人类免疫缺陷病毒1型（HIV）的新方法，但对于目前3400多万艾滋病毒感染者来说，它仍然是一种无法治愈的疾病，只能通过终身每日抗逆转录病毒治疗来治疗。因此，治愈艾滋病毒仍然是一个高度优先事项。潜伏的、具有复制能力的HIV在长期存活的CD4+ T细胞中无限期持续存在是实现治愈的主要障碍。目前正在开发针对这一潜在宿主的创新疗法，但需要更简单、更高通量和更精确的措施来加速进展。在本提案中，我们概述了一种研究策略，该策略将评估创新的细胞培养和HIV潜伏库的分子分析，这些分析具有比当前的“金标准”感染病毒恢复（IVR）分析更高通量、更高精度和更低成本的潜力。该提案的三个具体目标是：1)进一步开发和评估一种简化、高通量、精确和低成本的基于细胞培养的血液来源的静止CD4+ T细胞（rCD4细胞）的总诱导病毒恢复（TVR）试验，该试验可以在新鲜或冷冻保存的外周血单个核细胞（PBMC）上进行；2)比较使用从同一供体分离的rCD4细胞，Aim 1中开发的优化TVR试验的性能特征与使用HIV易感细胞系（Molt-4）而不是同种异体母细胞作为靶细胞的简化IVR试验的性能特征；3)评估TVR和IVR检测结果与HIV持久性分子测量之间的关系，包括增强的qPCR检测残余血浆病毒血症、细胞HIV RNA种类和rCD4细胞中HIV RNA与DNA比率，以确定潜伏但可诱导的HIV库的可靠分子替代品。目标1-3的完成将提供急需的工具来测量治疗干预后潜伏性HIV库的变化。拟议的工作将发展和评估细胞培养（可诱导病毒恢复）和潜伏库的分子分析。因此，该项目的目标与RFA-AI-13-038密切一致。简化分析的可用性

DESCRIPTION (provided by applicant): Novel approaches to prevent the acquisition of human immunodeficiency virus type 1 (HIV) by vaccination or pre-exposure prophylaxis are being vigorously pursued, but for the more than 34 million people currently living with HIV, it remains an incurable disease that can only be treated with lifelong daily antiretroviral therapy. As such, a cure for HIV remains a high priority. The indefinite persistence of latent, replication-competent HIV in long-lived CD4+ T cells is a major obstacle to achieving a cure. Innovative therapies are being developed to target this latent reservoir, but simpler, higher throughput and more precise measures are needed to accelerate progress. In this proposal, we outline a research strategy that will evaluate innovative cell culture and molecular assays of the latent reservoir of HIV that have the potential for higher throughput, greater precision and lower cost than the current "gold standard" assay of infectious virus recovery (IVR). The three specific aims of this proposal are: 1) to further develop and assess a simplified, high-throughput, precise and lower cost cell culture-based assay of total inducible virus recovery (TVR) from blood-derived resting CD4+ T cells (rCD4 cells) that can be performed on either fresh or cryopreserved peripheral blood mononuclear cells (PBMC); 2) to compare, using rCD4 cells isolated from the same donor, the performance characteristics of the optimized TVR assay developed in Aim 1 to that of a simplified IVR assay that uses an HIV susceptible cell line (Molt-4) rather than allogeneic blasts as target cells; and 3) to evaluate the relationships between TVR and IVR assay results and molecular measures of HIV persistence, including enhanced qPCR assays of residual plasma viremia, cellular HIV RNA species, and HIV RNA to DNA ratios in rCD4 cells to identify a reliable molecular surrogate of the latent but inducible HIV reservoir. Completion of Aims 1-3 should provide urgently needed tools to measure changes in the latent HIV reservoir following therapeutic interventions. The proposed work will develop and evaluate both cell culture (inducible virus recovery) and molecular assays of the latent reservoir. As such, the goals of this project are closely aligned with RFA-AI-13-038. The availability of simplified assays to quantify latent HIV will undoubtedly accelerate progress towards a cure by facilitating proof-of-concept studies of innovative therapies.