Quantification of the HIV-1 Reservoir by Immuno-PCR

 描述(由申请人提供)：消除或减少潜伏感染HIV-1的细胞池的治疗方法的开发需要可靠和可重复地评估潜伏感染池大小的标准化分析。建立这种检测方法面临三大挑战：i)潜伏感染细胞的频率非常低；ii)存在缺陷的细胞的频率远远高于复制能力强的前病毒；以及iii)缺乏潜伏感染细胞的独特生物标志物。到目前为止，已经使用了两种方法来估计潜在储集层的规模。基于聚合酶链式反应的检测方法以快速、灵敏和可重复的方式检测HIV-1DNA，但不能区分复制能力和缺陷前病毒，因此高估了病毒的储存库。相反，病毒生长分析(VOA‘s)确定了含有复制能力前病毒的细胞的频率，但它们耗费时间和劳动力，重复性差，并且不能捕获具有复制能力的前病毒的全部范围，导致对储存库的严重低估。我们的长期目标是开发一种技术，将PCR和QVOA的优势结合在一起，形成一种能够适应单细胞、高通量平台的检测方法。我们的中心假设是，免疫-聚合酶链式反应(IPCR)是使这一点成为可能的技术。IPCR的基本原理是用特定的抗体捕获感兴趣的抗原，然后用与报告DNA捆绑的第二个抗原特异性抗体进行检测，该DNA在利用荧光探针进行定量实时分析以及单细胞原位分析的聚合酶链式反应中被放大。该项目将受益于与Dr。 巴尔的摩人类病毒学研究所的尼尔·康斯坦丁开发并优化了iPCR技术。在具体目标1中，我们将开发一种vOA，其中p24的检测是通过iPCR进行的，显著提高了灵敏度、重复性和实际性能 这个化验结果。用iPCR检测病毒产量将与其他方法进行比较，这些方法估计潜在储存库的大小，包括通过与 HIV-1阴性献血者的淋巴母细胞。在具体目标2中，我们将开发高灵敏度的原位检测病毒抗原产生的iPCR技术和高通量流式细胞术分析技术。这种方法将允许对可以诱导产生病毒蛋白的单细胞进行鉴定和表型表征。最后，在具体目标3中，我们将评估iPCR能否在体外检测和体内临床试验中实现对潜伏期反转剂的病毒学效应的更敏感、更快速和更可重复性的检测，在临床试验中，潜伏期反转剂被应用于经ART治疗的HIV-1患者。完成该项目的目标将填补所需的技术空白 在根除试验中对潜伏宿主的准确估计，以及潜伏感染细胞的表型特征。

 DESCRIPTION (provided by applicant): The development of therapies to eliminate or reduce the pool of cells latently infected with HIV-1 requires standardized assays that reliably and reproducibly assess the size of the latent reservoir. Three major challenges face the establishment of such assay: i) the very low frequency of latently infected cells; ii) the much greater frequency of cells harboring a defective over replication competent provirus; and iii) the lack of distinctive biomarkers of latently infected cells. So far, two approaches have been used to estimate the size of the latent reservoir. PCR-based assays measure HIV-1 DNA in a rapid, sensitive, and reproducible fashion, but cannot discriminate between replication competent and defective proviruses, and thus overestimates the reservoir. By converse, viral outgrowth assays (VOA's) determine the frequency of cells harboring replication competent provirus, but they are time and labor intensive, poorly reproducible, and do not capture the full extent of replication competent proviruses, leading to a significant underestimation of the reservoir. Our long-term goal is to develop technologies that combine the advantages of PCR with those of the QVOA into an assay that can be adapted to a single-cell, high-throughput platform. Our central hypothesis is that immune-PCR (iPCR) is the technology that makes that possible. The underlying principle of iPCR is the capture of an antigen of interest with a specific antibody, followed by detection with a second antigen-specific antibody tethered to a reporter DNA that is amplified in PCR reactions employing fluorescent probes for quantitative real-time analyses, as well as for single-cell in situ analyses. This project will benefit from the collaboration with Dr. Niel Constantine at the Institute of Human Virology in Baltimore, who developed and optimized the iPCR technology. In Specific Aim 1, we will develop a VOA where detection of p24 is carried out by iPCR, significantly improving sensitivity, reproducibility and practical performance of this assay. Detection of viral production by iPCR will be compared to other methods that estimate the size of the latent reservoir, both with and without viral expansion by co-culture with lymphoblasts of HIV-1 negative donors. In Specific Aim 2, we will develop techniques for ultrasensitive in situ detection of viral antigen production by iPCR and analysis by high-throughput flow cytometry. This approach will allow the identification and phenotypic characterization of single cells in which production of viral protein can be induced. Finally, in Specific Aim 3, we will evaluate whether iPCR can be used to achieve a more sensitive, rapid and reproducible detection of virologic effects of latency-reversing agents in ex vivo assays, and in vivo in clinical trials in which latency-reversing agents are administered to ART-treated HIV-1 patients. Accomplishing the goals of this project will fill the technological gap needed to obtain an accurate estimate of the latent reservoir in eradication trials, and a phenotypic characterization of latently infected cells.