Simplified Assays of Latent But Inducible HIV-1 Reservoirs

描述(申请人提供)：正在积极寻求通过接种疫苗或暴露前预防措施来防止感染人类免疫缺陷病毒1型(HIV)的新方法，但对于目前感染艾滋病毒的3400多万人来说，这仍然是一种无法治愈的疾病，只能通过终生每日抗逆转录病毒疗法进行治疗。因此，治愈艾滋病毒仍然是一个高度优先的问题。潜伏的、具有复制能力的艾滋病毒在长期存活的CD4+T细胞中无限期地持续存在，是实现治愈的主要障碍。正在开发针对这一潜在储藏者的创新疗法，但需要更简单、更高的产量和更精确的措施来加快进展。在这项提案中，我们概述了一种研究战略，该战略将评估创新的HIV潜伏库细胞培养和分子分析，这些方法具有比目前感染病毒恢复(IVR)的“金标准”分析更高的吞吐量、更高的精确度和更低的成本。这项建议的三个具体目标是：1)进一步发展和评估一种基于细胞培养的简化、高通量、精确和低成本的从血液来源的静止CD4+T细胞(rCD4细胞)中总可诱导病毒回收率(TVR)的检测方法，该方法可以在新鲜或冷冻保存的外周血单个核细胞(PBMC)上进行；2)使用从同一供体分离的rCD4细胞，比较在目标1中开发的优化的TVR检测与使用HIV敏感细胞系(Molt-4)而不是同种异基因母细胞作为靶细胞的简化IVR检测的性能特征；3)评估TVR和IVR检测结果与HIV持久性的分子指标之间的关系，包括增强的血浆残留病毒血症、细胞内HIV RNA种类和rCD4细胞中HIV RNA/DNA比率的qPCR分析，以确定潜在但可诱导的HIV储备库的可靠分子替代物。目标1-3的完成应提供急需的工具，以衡量治疗干预后艾滋病毒潜伏宿主的变化。这项拟议的工作将开发和评估细胞培养(可诱导病毒恢复)和潜在储存库的分子分析。因此，本项目的目标与RFA-AI-13-038密切相关。简化化验的可用性 量化潜伏的艾滋病毒无疑将通过促进创新疗法的概念验证研究来加速治愈的进展。

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.