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: 8885651
• 负责人: John W Mellors
• 金额: $ 22.89万
• 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 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8885651
• 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; Health; 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; 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）：正在积极寻求通过疫苗接种或暴露前预防来预防感染 1 型人类免疫缺陷病毒 (HIV) 的新方法，但对于目前超过 3400 万艾滋病毒感染者来说，这仍然是一个难题。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当务之急。潜伏的、具有复制能力的 HIV 在长寿命 CD4+ T 细胞中无限期存留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、更高的通量和更精确的措施来加速进展。在本提案中，我们概述了一项研究策略，该策略将评估 HIV 潜伏库的创新细胞培养和分子测定，与目前感染性病毒回收的“金标准”测定相比，这些方法具有更高的通量、更高的精度和更低的成本（交互式语音应答）。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、精确且成本较低的基于细胞培养的检测方法，用于从血源性静息 CD4+ T 细胞 (rCD4) 中检测总诱导病毒回收率 (TVR)细胞），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 rCD4 细胞，将目标 1 中开发的优化 TVR 测定的性能特征与使用 HIV 易感细胞系 (Molt-4) 而不是同种异体母细胞的简化 IVR 测定的性能特征进行比较靶细胞； 3) 评估TVR和IVR检测结果与HIV持续性分子测量之间的关系，包括对残余血浆病毒血症、细胞HIV RNA种类和rCD4细胞中HIV RNA与DNA比率的增强qPCR检测，以确定可靠的分子替代物潜伏但可诱导的艾滋病毒储存库。目标 1-3 的完成应提供急需的工具来测量治疗干预后潜在 HIV 病毒库的变化。拟议的工作将开发和评估细胞培养（诱导病毒回收）和潜伏病毒库的分子测定。因此，该项目的目标与 RFA-AI-13-038 密切一致。简化检测的可用性 量化潜在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。