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

描述(申请人提供)：HIV-1在病毒库中的持久性一直是根除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，被发现为残留的低水平病毒血症(LLV)，静息记忆CD4+T细胞的潜伏感染得到了很好的证实。已经开发出一种方法来量化接受HAART的患者的静息CD4+T细胞中含有复制能力HIV-1的细胞。病毒生长试验是目前测量潜伏感染HIV-1的静止CD4+T细胞的金标准方法。然而，这种检测方法需要从血液中提纯大量静止的CD4+T细胞，而且需要进行一系列稀释才能进行检测，而且成本高、劳动强度大 每百万感染单位(IUPM)静息的CD4+T细胞。为了克服当前检测的问题，在本应用中，我们提出了一种基于下一代测序(NGS)技术的检测方法。在建议的检测中，将直接分析从静止的CD4+T细胞批量培养的不同病毒序列的数量，以计分潜伏感染HIV-1的CD4+T细胞的数量。由于深度测序对检测病毒种群中的多个变异高度敏感，因此可以计算在批量培养中被诱导复制的不同病毒的数量。这些研究将为治愈HIV感染提供有价值的信息，并导致开发一种能够以更高的准确性、更少的时间和资源消耗以及更少的劳动密集型来量化潜伏的HIV-1储存库的方法。

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.