Simplified Assays of Latent But Inducible HIV-1 Reservoirs

潜在但可诱导的 HIV-1 储库的简化检测

• 批准号: 8766909
• 负责人: John W Mellors
• 金额: $ 20.39万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-03 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8766909
• 关键词: Allogenic; Biological; Biological Assay; Blast Cell; Blood; Blood Volume; CD4 Positive T Lymphocytes; Cell Culture Techniques; Cell Line; Cells; Characteristics; Clinical Research; DNA; Data; Data Set; Disease; Genome; Goals; Gold; HIV; HIV-1; Head; Human; Immunologic Deficiency Syndromes; Innovative Therapy; Integrase; Intervention; Life; Measures; Molecular; Molecular Analysis; Molting; National Institute of Allergy and Infectious Disease; Patients; Performance; Peripheral Blood Mononuclear Cell; Plasma; Prophylactic treatment; RNA; Recovery; Research; Residual state; Rest; Sampling; Signal Transduction; Speed; Surrogate Markers; Therapeutic Intervention; Vaccination; Variant; Viral; Viremia; Virus; Work; antiretroviral therapy; base; cost; head-to-head comparison; improved; in vivo; innovation; miniaturize; novel strategies; prevent; prototype; public health relevance; tool

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.

描述（由申请人提供）：通过接种疫苗或暴露前预防来预防人类免疫缺陷病毒1型（HIV）感染的新方法正在大力推行，但对于目前感染HIV的3400多万人来说，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、有复制能力的HIV在长寿的CD 4 + T细胞中的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储库的创新疗法，但需要更简单、更高通量和更精确的措施来加速进展。在这项提案中，我们概述了一项研究战略，将评估创新的细胞培养和HIV潜伏库的分子检测，这些检测具有比目前的感染性病毒恢复（IVR）“金标准”检测更高的通量，更高的精度和更低的成本的潜力。本研究的三个具体目标是：1）进一步开发和评估一种简化的、高通量的、精确的和低成本的基于细胞培养的从血液来源的静息CD 4 + T细胞中回收总诱导病毒（TVR）的检测方法（rCD 4细胞），其可以在新鲜或冷冻保存的外周血单核细胞（PBMC）上进行; 2）使用从同一供体分离的rCD 4细胞，比较目标1中开发的优化TVR测定与使用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测定的性能特征;和3）评价TVR和IVR检测结果与HIV持久性的分子指标之间的关系，包括残留血浆病毒血症、细胞HIV RNA种类和rCD 4细胞中HIV RNA与DNA比率的增强qPCR检测，以鉴定潜伏但可诱导的HIV储库的可靠分子替代物。目标1-3的完成应提供迫切需要的工具，以衡量治疗干预后潜伏的艾滋病毒库的变化。拟议的工作将开发和评估细胞培养（诱导型病毒回收）和潜在水库的分子测定。因此，本项目的目标与RFA-AI-13-038密切一致。简化分析的可用性 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。