A Novel Microfluidic HIV-1 Co-Culture Assay to Quantify Latent Reservoirs

一种新颖的微流体 HIV-1 共培养测定法来量化潜在储库

• 批准号: 8768888
• 负责人: Utkan Demirci
• 金额: $ 22.62万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8768888
• 关键词: 3-Dimensional; Activated Lymphocyte; Allogenic; Anti-Retroviral Agents; Automation; Biological Assay; Biological Markers; Biomedical Engineering; Biophysics; Blast Cell; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Communication; Cell Culture Techniques; Cells; Chromosomes; Coculture Techniques; Code; Complex; Coupled; Culture Media; DNA; Detection; Development; Disease remission; Frequencies; Genetic Materials; Goals; Grant; Growth; HIV; HIV-1; Human; Individual; Infection; Laboratories; Laboratory Study; Measurement; Measures; Methods; Microfluidics; Modeling; Organ; Patients; Performance; Peripheral Blood Mononuclear Cell; Principal Investigator; Proteins; Protocols documentation; Proviruses; Reaction; Reagent; Research; Rest; Retrovirology; Sampling; Stem cell transplant; System; Technology; Test Result; Testing; Therapeutic; Time; Tissues; Toxin; Variant; Viral; Viral Genome; Virus; Virus-Cell Membrane Interaction; antiretroviral therapy; base; cell type; chemotherapy; cohort; design; experience; improved; in vivo; innovation; miniaturize; novel; public health relevance; research study; response; viral DNA; viral RNA; virology

DESCRIPTION (provided by applicant): HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy, and a main challenge in achieving HIV-1 antiretroviral (ART)-free remission is the persistence of these latent viral reservoirs. Quantifying the replication competent viral reservoir is critical to understanding how HIV-1 persists and to measuring how this reservoir changes in response to therapeutic strategies. Quantitative co-culture assay have been developed to measure the replication competent reservoir by activating CD4+ T cells from patients in the presence of feeder cells for viral outgrowth and measurements of HIV-1 proteins or genetic material. However, traditional quantitative co-culture assays are time-consuming, labor-intensive and require parallel reactions in large-well format culture. Novel methods to miniaturize the co- culture platform and to increase sample throughput have the potential significantly to improve the study of HIV latency and could be used to streamline and standardize testing of novel reservoir eradication strategies. We propose to adapt and validate a novel, miniaturized chip-based microfluidic co-culture assay and compare its performance with the traditional co-culture assay. We hypothesize that miniaturizing these assays will allow for higher throughput and be less labor intensive than traditional platforms. The proposed method will also allow for tight control of the growth microenvironment which will help to improve sensitivity and standardize test results between laboratories. Our aims are: (1) adapt a chip-based microfluidic cell culture/expansion protocol for viral co-culture to quantify integrated, replication-competent provirus as infectious units per million cells, and, (2) validate the performance of the novel microfluidic co-culture assay compared with traditional laboratory viral outgrowth assays. This two-year development grant will utilize innovative approaches and adaptations of existing microfluidic technologies to develop an assay to characterize HIV- reservoirs in patients on antiretroviral therapy. Our proposal involves principal investigators with different but complimentary research backgrounds and experiences, including translational virology and bioengineering/biophysics. Our proposed assay has the potential to be used for a variety of applications involving the isolation and growth of infected human cell subsets and tissues, and we ultimately plan to use the assay to quantify replication competent reservoirs in various cell types and from patients undergoing allogeneic stem cell transplantation and cytoreductive chemotherapy.

描述(申请人提供)：尽管长期使用抗逆转录病毒疗法抑制循环病毒，但艾滋病毒-1储存库仍然以潜伏的形式存在，实现艾滋病毒-1抗逆转录病毒(ART)无缓解的主要挑战是这些潜伏病毒储存库的持久性。量化具有复制能力的病毒库对于了解HIV-1如何持续存在和测量该病毒库如何随着治疗策略的反应而变化至关重要。已开发出定量共培养试验来测量复制能力，方法是在有滋养细胞存在的情况下激活患者的CD4+T细胞，用于病毒生长和HIV-1蛋白质或遗传物质的测量。然而，传统的定量共培养分析是耗时、劳动密集型的，并且需要在大孔培养中进行平行反应。将共培养平台小型化和增加样本吞吐量的新方法有可能显著改善艾滋病毒潜伏期的研究，并可用于简化和标准化新的水库根除策略的测试。我们建议修改和验证一种新颖的、微型化的基于芯片的微流控共培养方法，并将其性能与传统的共培养方法进行比较。我们假设，与传统平台相比，将这些分析小型化将允许更高的吞吐量和更少的劳动密集型。拟议的方法还将允许严格控制生长微环境，这将有助于提高敏感度并使实验室之间的测试结果标准化。我们的目标是：(1)采用基于芯片的微流控细胞培养/扩增方案用于病毒共培养，以量化整合的、具有复制能力的前病毒作为每百万细胞的感染单位，(2)验证新型微流控共培养方法与传统实验室病毒生长检测方法的性能。这笔为期两年的开发赠款将利用现有微流控技术的创新方法和改编，开发一种分析方法，以确定接受抗逆转录病毒治疗的患者中艾滋病毒宿主的特征。我们的建议涉及具有不同但互补的研究背景和经验的主要研究人员，包括翻译病毒学和生物工程/生物物理学。我们提出的检测方法有可能用于各种应用，包括分离和生长受感染的人类细胞亚群和组织，我们最终计划使用该检测方法来量化各种细胞类型和接受异基因干细胞移植和细胞减少性化疗的患者的复制能力储存库。