Quantification of the HIV-1 Reservoir by Immuno-PCR

 描述（由申请人提供）：开发消除或减少潜伏感染HIV-1的细胞池的治疗方法需要标准化检测，以可靠和可重复地评估潜伏库的大小。建立这种测定法面临三个主要挑战：i）潜伏感染细胞的频率非常低; ii）携带有缺陷的过度复制能力前病毒的细胞的频率高得多;和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层的大小。基于PCR的检测以快速、灵敏和可重复的方式测量HIV-1 DNA，但不能区分有复制能力的前病毒和有缺陷的前病毒，因此高估了储存库。匡威，病毒生长测定（VOA）确定携带有复制能力的前病毒的细胞的频率，但它们是时间和劳动密集型的，可再现性差，并且不能捕获复制能力的前病毒的全部范围，导致对储库的显著低估。 我们的长期目标是开发一种技术，将PCR和QVOA的优点联合收割机结合到一种适用于单细胞、高通量平台的检测方法中。我们的核心假设是免疫PCR（iPCR）是使这成为可能的技术。iPCR的基本原理是用特异性抗体捕获感兴趣的抗原，然后用与报告DNA连接的第二抗原特异性抗体进行检测，所述报告DNA在PCR反应中使用荧光探针扩增，用于定量实时分析以及单细胞原位分析。该项目将受益于与博士的合作。 开发并优化iPCR技术的巴尔的摩人类病毒学研究所的Niel Constantine说。 在具体目标1中，我们将开发一种VOA，通过iPCR检测p24，显著提高灵敏度、重现性和实用性能 这个化验。通过iPCR检测病毒产生将与估计潜伏储库大小的其他方法进行比较，无论是否通过与以下物质共培养进行病毒扩增： HIV-1阴性供体的淋巴母细胞。 在特定目标2中，我们将开发通过iPCR进行病毒抗原生产的超灵敏原位检测和通过高通量流式细胞术进行分析的技术。该方法将允许鉴定和表型表征其中可以诱导病毒蛋白质产生的单细胞。 最后，在具体目标3中，我们将评估iPCR是否可用于在离体试验中以及在ART治疗的HIV-1患者接受潜伏逆转剂给药的临床试验中实现潜伏逆转剂病毒学效应的更灵敏、快速和可重现的检测。 实现该项目的目标将填补技术空白， 在根除试验中对潜伏库的准确估计，以及潜伏感染细胞的表型表征。

 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.