New and Simple Assay to Measure Latent HIV Reservoir Using Deep Sequencing Method

使用深度测序方法测量潜在 HIV 病毒库的新且简单的检测方法

• 批准号: 8768898
• 负责人: Sook-Kyung Lee
• 金额: $ 22.8万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8768898
• 关键词: Biological Assay; Blood; CD4 Positive T Lymphocytes; Cell fusion; Cells; Chronic; Development; Ensure; Enzyme-Linked Immunosorbent Assay; Gene Expression; Gold; HIV; HIV Infections; HIV-1; HIV-2; Highly Active Antiretroviral Therapy; Infection; Interphase Cell; Lead; Measurement; Measures; Mediating; Methods; Minor; Nucleotides; Patients; Polyethylene Glycols; Population; Procedures; Protocols documentation; Proviruses; Records; Residual state; Resources; Rest; Sampling; Sequence Analysis; Source; System; T-Cell Activation; T-Lymphocyte; Technology; Time; Variant; Viral; Viral Genes; Viremia; Virus; Work; assay development; base; cell transformation; cost effective; deep sequencing; env Gene Products; improved; latent infection; memory CD4 T lymphocyte; next generation sequencing; public health relevance; tat Protein; tool

DESCRIPTION (provided by applicant): HIV-1 persistence in viral reservoirs has been the major obstacle for the eradication of HIV-1. Latently infected T cells are one of the potential sources of the virus that is found as residual low-level viremia (LLV) and latent infection of resting memory CD4+ T cells is well demonstrated. Assays have been developed to quantify cells that harbor replication-competent HIV-1 among resting CD4+ T cells in patients on HAART. A viral outgrowth assay is the current gold-standard method to measure latently HIV-1 infected resting CD4+ T cells. However, this assay requires large numbers of resting CD4+ T cells purified from blood and is costly and labor- intensive involving a serial dilution to measure infectious units per million (IUPM) resting CD4+ T cells. To overcome the issues with the current assay, in this application we propose an assay based on next- generation sequencing (NGS) technology. In the proposed assay, the number of different sequences of the viruses bulk-cultured from the resting CD4+ T cells will be directly analyzed to score the number of latently HIV-1-infected CD4+ T cells. Since deep sequencing is highly sensitive to detect multiple variants in a viral population, it will be possible to count the number of distinct viruses that hae been induced to replicate in a bulk culture. These studies will provide valuable information for curing HIV infection and lead to a development of an assay to quantify the latent HIV-1 reservoir with improved accuracy, less time and resource consuming, and less labor intensive.

描述（由申请人提供）：HIV-1在病毒储库中的持续存在一直是根除HIV-1的主要障碍。潜伏感染的T细胞是病毒的潜在来源之一，其被发现为残留的低水平病毒血症（LLV），并且静息记忆CD 4 + T细胞的潜伏感染被充分证明。已经开发了检测方法来定量HAART患者静息CD 4 + T细胞中携带有复制能力的HIV-1的细胞。病毒生长试验是目前测量潜伏HIV-1感染的静息CD 4 + T细胞的金标准方法。然而，该测定需要大量从血液中纯化的静息CD 4 + T细胞，并且昂贵且劳动密集，涉及连续稀释以测量 感染单位/百万（IUPM）静息CD 4 + T细胞。为了克服当前测定的问题，在本申请中，我们提出了基于下一代测序（NGS）技术的测定。在拟定的试验中，将直接分析从静息CD 4 + T细胞中批量培养的病毒的不同序列的数量，以对潜伏HIV-1感染的CD 4 + T细胞的数量进行评分。由于深度测序对检测病毒群体中的多种变异非常敏感，因此有可能对大量培养物中被诱导复制的不同病毒的数量进行计数。这些研究将为治疗HIV感染提供有价值的信息，并导致开发一种测定方法，以提高准确性，减少时间和资源消耗，减少劳动强度来量化潜伏的HIV-1储库。