Quantitative Viral Outgrowth Assays with Improved Throughput and Performance

提高通量和性能的定量病毒生长检测

基本信息

批准号：
8790227
负责人：
CHRISTOS J PETROPOULOS
金额：
$ 15万
依托单位：
MONOGRAM BIOSCIENCES, INC.
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：
2014-07-15 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8790227
关键词：
Acquired Immunodeficiency Syndrome Address Affect Anti-Retroviral Agents Antibodies Antigens Apoptosis Automation Basic Science Biological Assay Blood Blood Cells Blood donor Blood specimen CD4 Positive T Lymphocytes Cell Count Cell Culture Techniques Cell Fraction Cell Separation Cells Cessation of life Clinical Clinical Investigator Clinical Trials Collaborations Collection Consensus Culture Media DNA Data Set Detection Development Devices Disease remission Effectiveness Enzyme-Linked Immunosorbent Assay Evaluation Exposure to Ficoll Funding Funding Opportunities Future Gene Expression Goals Gold HIV HIV Infections HIV vaccine HIV-1 Harvest Histone Deacetylase Inhibitor Individual Infection Laboratories Latent Virus Letters Leukapheresis Literature Lymphocyte Magnetism Measurement Measures Messenger RNA Methods Molecular Nanotechnology Performance Peripheral Blood Mononuclear Cell Pharmaceutical Preparations Plasma Population Procedures Process Production Proteins Protocols documentation Proviruses Publishing Regimen Reproducibility Residual state Resources Rest Reverse Transcriptase Polymerase Chain Reaction Sampling Specific qualifier value T-Cell Activation T-Lymphocyte Technology Testing Therapeutic Time Tissues Validation Variant Vial device Viral Virion Virus Virus Activation assay development base cell type chromatin remodeling clinically relevant comparative design experience gp160 immune activation improved in vivo innovation model development monocyte neutralizing antibody novel peripheral blood prevent public health relevance vaccination strategy vaccine candidate viral DNA

项目摘要

DESCRIPTION (provided by applicant): Strategies to eradicate HIV-1 infection in individuals with suppressed or undetectable viral replication are being actively explored in clinical trials. HIV+ individuals with suppressed viral replication are treated with agents that remodel chromatin, e.g. histone deacetylase inhibitors (HDACIs) or other T cell stimulators in efforts to reactivate expression of latent HIV resulting in de novo virus production, which will subsequently results in the death of latent infected cells through a variety of postulated mechanisms, including programmed cell death (apoptosis) and/or immune activation. New virions produced by activated T cells are prevented from infecting new cells by the ongoing treatment of the individual with a fully suppressive anti-retroviral drug regimen. Such virus elimination strategies are predicated on the assumption that multiple rounds of treatment with latency reversing drugs will incrementally diminish the latent vial reservoir over time leading to an eventual eradication of infection. A major challenge in evaluating the efficacy of eradication protocols in clinical trils is the lack of a standardized assay for detecting latent virus depletion as a measure of reservoir eradication. The current recognized gold-standard assay (Q-VOA) is based on quantitative measures of viral outgrowth using limit dilutions of primary resting T cells isolated following exposure to candidate therapeutic T cell stimulators and potent control compounds. These assays require large cell numbers and require days, if not weeks to complete. Virus production is quantitated from cell or cell culture media fractions using various methods that include ELISA and PCR. The use of various assays and detection methods has led to inconsistent results from the various performing laboratories and hinders inter-laboratory comparisons. Previously, the introduction of standardized neutralizing antibody assays for the evaluation of HIV vaccine candidates enabled fine distinctions in efficacy when evaluating different immunogens and vaccination strategies. In much the same way, the implementation of a reliable, standardized Q-VOA assay should provide the ability to discern subtle differences in latent reservoir size during eradication studies. Monogram Biosciences (www.monogrambio.com) has more than 15 years of experience developing high throughput cell based viral assays that evaluate HIV-1 therapeutics. We intend to leverage our established expertise to pursue and complete the proposed study objectives. Monogram will explore different Q-VOA approaches to select and develop a method that generates a robust, reproducible, rapid and affordable method to accurately measure the HIV-1 latent reservoir in clinical samples. This study proposal addresses several specific objectives as specified in the funding opportunity announcement (PA-12-162): (a) Development of new assays (including but not limited to development of new quantitative assays for sensitive detection of HIV-1 in tissue, a simple method for detecting replication-competent virus in latently infected cells, assays to measure diversification of viruse in reservoirs, assays to accurately discriminate and measure vDNA in integrated and unintegrated forms. (b) Technology advancement (including but not limited to methods to standardize isolation and quantification of replication competent vRNA and viral DNA (vDNA) from cells and tissues, and nanotechnology).

描述（由申请人提供）：在临床试验中正在积极探索抑制或无法检测到的病毒复制患者中HIV-1感染的策略。 HIV+患有抑制病毒复制的个体用重塑染色质的药物治疗，例如组蛋白脱乙酰基酶抑制剂（HDACIS）或其他T细胞刺激剂，努力重新激活潜在的HIV表达，从而导致从头病毒产生，这将随后通过包括多种假设的机制导致潜在感染细胞死亡程序性细胞死亡（凋亡）和/或免疫激活。活化的T细胞产生的新病毒体可通过持续治疗完全抑制性抗返回药物方案的个体来感染新细胞。消除这种病毒策略基于以下假设：随着时间的流逝，多发药物的多轮治疗将逐渐减少潜在的小瓶储层，从而导致最终消除感染。评估根除方案在临床三元素中的疗效的主要挑战是缺乏用于检测潜在病毒消耗的标准化测定，以衡量消除储层的量度。当前公认的黄金标准测定法（Q-VOA）基于病毒生长的定量度量，使用了暴露于候选治疗性T细胞刺激剂和有效的控制化合物后分离的原代静息T细胞的极限稀释液。这些测定需要大的单元格数，并且需要数天（如果不是几周才能完成）。使用包括ELISA和PCR在内的各种方法，从细胞或细胞培养基的分数中定量病毒的产生。各种测定和检测方法的使用导致各种表现实验室和阻碍实验室间比较的结果不一致。以前，在评估不同的免疫原子和疫苗接种策略时，引入了评估HIV疫苗候选物的标准化中和抗体测定。以几乎相同的方式，实施可靠的，标准化的Q-VOA分析应提供消除研究期间潜在储层大小的细微差异的能力。 Monogram Biosciences（www.mongragmbio.com）具有15年以上的经验，发展了评估HIV-1治疗剂的高通量细胞病毒测定法。我们打算利用我们既定的专业知识来追求和完成拟议的研究目标。 Monogram将探索不同的Q-VOA方法，以选择和开发一种产生健壮，可重现，快速和负担得起的方法，以准确测量临床样品中的HIV-1潜在储层。 This study proposal addresses several specific objectives as specified in the funding opportunity announcement (PA-12-162): (a) Development of new assays (including but not limited to development of new quantitative assays for sensitive detection of HIV-1 in tissue, a simple method for detecting replication-competent virus in latently infected cells, assays to measure diversification of viruse in reservoirs, assays to accurately discriminate and measure vDNA in综合和不整合的形式（b）技术的进步（包括但不限于从细胞和组织中隔离和量化复制的隔离和量化方法，以及纳米技术）。