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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：尽管通过抗逆转录病毒治疗长期抑制了循环病毒，HIV-1 储存库仍然以潜伏形式存在，实现无 HIV-1 抗逆转录病毒 (ART) 缓解的主要挑战是这些储存库的持续存在潜伏病毒库。量化具有复制能力的病毒库对于了解 HIV-1 如何持续存在以及测量该病毒库如何响应治疗策略而变化至关重要。定量共培养测定已被开发出来，通过在存在饲养细胞的情况下激活患者的 CD4+ T 细胞以进行病毒生长，并测量 HIV-1 蛋白或遗传物质，从而测量复制能力储存库。然而，传统的定量共培养测定耗时、劳动密集，并且需要在大孔格式培养中进行平行反应。使共培养平台小型化并提高样品通量的新方法有可能显着改善 HIV 潜伏期的研究，并可用于简化和标准化新型储存库根除策略的测试。我们建议采用和验证一种新型的、小型化的基于芯片的微流体共培养测定，并将其性能与传统的共培养测定进行比较。我们假设，与传统平台相比，小型化这些测定将允许更高的通量并减少劳动力密集度。所提出的方法还将允许严格控制生长微环境，这将有助于提高灵敏度并标准化实验室之间的测试结果。我们的目标是：（1）采用基于芯片的微流控细胞培养/扩增方案进行病毒共培养，以将集成的、具有复制能力的原病毒量化为每百万细胞的感染单位，以及（2）验证新型微流控的性能共培养测定与传统实验室病毒生长测定的比较。这笔为期两年的开发补助金将利用创新方法和现有微流体技术的改进来开发一种测定方法，以表征接受抗逆转录病毒治疗的患者中的 HIV 病毒库。我们的提案涉及具有不同但互补的研究背景和经验的主要研究人员，包括转化病毒学和生物工程/生物物理学。我们提出的测定法有可能用于各种应用，包括受感染的人类细胞亚群和组织的分离和生长，我们最终计划使用该测定法来量化各种细胞类型和接受同种异体干细胞治疗的患者中的复制能力储存库细胞移植和细胞减灭化疗。