The Dual Reporter Sensor Cell (DRSC) Assay: An Enhanced Tool for Measuring the Viral Reservoir

双报告传感器细胞 (DRSC) 检测：测量病毒库的增强工具

• 批准号: 10254314
• 负责人: Scott M Berry
• 金额: $ 18.73万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-04 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10254314
• 关键词: APOCEC3G gene; Address; Antiviral Agents; Antiviral Therapy; Behavior; Biological Assay; Blood; CD4 Positive T Lymphocytes; Cell Line; Cells; Cellular Assay; Chemicals; Clinical; Complex; Convection; Cultured Cells; Custom; DNA; Data; Development; Devices; Diffusion; Effectiveness; Environment; Event; Foundations; Future; Genotype; Geometry; Gold; HIV; HIV Genome; HIV Infections; HIV-1; Half-Life; Image; Image Analysis; Infection; Kinetics; Link; Liquid substance; Measurement; Measures; Mechanics; Mediation; Modification; Monitor; Nucleic Acids; Patients; Performance; Phase; Pilot Projects; Plaque Assay; Population; Process; Production; Proteins; Protocols documentation; Reporter; Research; Resolution; Rest; Role; Sampling; Scanning; Signal Transduction; Specificity; System; T-Lymphocyte; Techniques; Technology; Therapeutic; Time; Translations; Universities; Validation; Viral; Viral reservoir; Viremia; Virion; Virus; Virus Latency; Wisconsin; assay development; automated image analysis; base; clinical implementation; improved; latent infection; next generation; novel; phenotypic data; prospective; real-time images; research study; response; sensor; tool

PROJECT SUMMARY The latent viral reservoir is a major barrier to eradicating HIV infection. Unfortunately, very few tools are available to quantify the reservoir and monitor the effectiveness of prospective therapies that aim to reduce it. The current gold standard assay, the quantitative viral outgrowth assay (QVOA), has enabled landmark scientific studies, but lacks the simplicity and robustness required for large-scale clinical implementation. Other options include a number of nucleic acid-based assays, but these are thought to overestimate the quantity of latent HIV that is actually replication-competent. Thus, there exists an unmet need for next-generation reservoir assay technologies that are capable of analyzing the HIV proviral reservoir in a straightforward and precise manner. The cornerstone of our proposed assay is a novel “dual reporter” sensor cell (DRSC) line that contains two independent HIV infection-linked reporter systems, both of which must be triggered to designate a “true positive” result, thereby enhancing specificity. Like QVOA, our DRSC requires successful spread of HIV infection between multiple cells to trigger a positive signal, thereby eliminating many of the false positive events seen with non-live cell assays (e.g., defective HIV DNA will be erroneously quantified in many PCR-based reservoir assay). Importantly, preliminary data suggests that the DRSC is significantly faster (2 vs. 7 days) and requires less sample volume when compared with the QVOA. In the proposed project, we further optimize the DRSC via development of two key concepts: First, we will evaluate and implement “low convection” culture environments, which will accelerate assay kinetics (and improve sensitivity) via elimination of the convective dilution of secreted factors (including virions), resulting in the concentration of factors within the vicinity of producing cells. Second, we will continue to improve our automated imaging and image analysis techniques (including real-time imaging processes), facilitating the further optimization of assay performance (e.g., through better selection of assay timing) and streamlining the ultimate translation of this technology into broader research and clinical roles (e.g., via integration of “image-based genotyping” into our readout). These two key developments enable a number of advantageous features, including: 1) Reduction in total assay time; 2) The spatial resolution of the DRSCs combined with diffusion- dominant fluid mechanics eliminates the need for limiting dilution endpoints (as with QVOA, which requires multiple replicates of multiple T cell dilutions to obtain a single endpoint), substantially reducing the amount of sample volume required; 3) an overall reduction in assay complexity, relative to the QVOA. Importantly, at the conclusion of this study, we will perform a pilot study comparing patient samples with expected high and low reservoir values using both the DRSC assay and QVOA, thus laying the foundation for larger future studies.

项目摘要 潜伏的病毒库是根除艾滋病毒感染的主要障碍。不幸的是， 量化水库，并监测旨在减少水库的前瞻性疗法的有效性。目前， 金标准测定法，即定量病毒生长测定法（QVOA），已经使具有里程碑意义的科学研究成为可能， 但缺乏大规模临床实施所需的简单性和鲁棒性。其他选项包括 许多基于核酸的检测，但这些被认为高估了潜伏的艾滋病毒的数量， 实际上是可以复制的。因此，存在对下一代储层测定的未满足的需求 能够以直接和精确的方式分析HIV前病毒库的技术。 我们提出的测定的基石是一种新型的“双报告”传感器细胞（DRSC）系，其含有两个 独立的HIV感染相关报告系统，必须触发这两个系统才能指定“真阳性” 结果，从而提高特异性。与QVOA一样，我们的DRSC要求艾滋病毒感染在 多个细胞来触发阳性信号，从而消除了非活细胞中观察到的许多假阳性事件。 细胞测定（例如，在许多基于PCR的储库测定中，缺陷HIV DNA将被错误地定量）。 重要的是，初步数据表明，DRSC明显更快（2对7天），需要更少 与QVOA相比，样品体积。 在建议的项目中，我们通过两个关键概念的发展进一步优化DRSC：首先，我们将 评估和实施“低对流”培养环境，这将加速测定动力学（并改善 通过消除分泌因子（包括病毒体）的对流稀释， 生产细胞附近的因子浓度。其次，我们将继续改进我们的自动化 成像和图像分析技术（包括实时成像过程），促进进一步的 测定性能的优化（例如，通过更好地选择测定时间）和简化最终的 将该技术转化为更广泛的研究和临床角色（例如，通过集成“基于图像的 基因分型”到我们的读数中）。这两个关键的发展实现了许多有利的特征， 包括：1）总测定时间的减少; 2）结合扩散的DRSC的空间分辨率- 主导流体力学消除了对有限稀释终点的需要（与QVOA一样，QVOA需要 多个T细胞稀释液的多个重复以获得单个终点），实质上减少了T细胞的量。 所需的样品体积; 3）相对于QVOA，测定复杂性的总体降低。重要的是，在 本研究的结论，我们将进行一项初步研究，比较患者样本与预期的高和低 利用DRSC分析和QVOA分析确定了储集层价值，从而为今后更大规模的研究奠定了基础。