A Novel Influenza Neuraminidase Inhibition Assay

一种新型流感神经氨酸酶抑制试验

• 批准号: 7666355
• 负责人: XING-XIANG LI
• 金额: $ 27.1万
• 依托单位: CELLEX, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2009-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7666355
• 关键词: Antiviral Agents; Biochemical; Biological Assay; Cell Culture Techniques; Cellular Assay; Chemiluminescence assay; Cleaved cell; Clinical; Computer Simulation; Data; Detection; Development; Drug Formulations; Drug Utilization Review; Drug resistance; Drug usage; Evaluation; Exhibits; Fluorescence; Fluorogenic Substrate; Generations; Goals; Half-Life; High Pressure Liquid Chromatography; Hour; Human; Influenza; Influenza A Virus, H5N1 Subtype; Influenza B virus; Inhibitory Concentration 50; Intervention; Life Cycle Stages; Light; Luciferases; Manuals; Measures; Methods; Monitor; Mutation; Nasal Lavage Fluid; Neuraminic Acids; Neuraminidase; Neuraminidase inhibitor; One-Step dentin bonding system; Operative Surgical Procedures; Oseltamivir; Performance; Pharmaceutical Preparations; Phase; Play; Predisposition; Preparation; Procedures; Protocols documentation; Reaction; Reagent; Recording of previous events; Reproducibility; Research; Resistance; Role; Sampling; Sewage; Signal Transduction; Small Business Innovation Research Grant; Specimen; Surveillance Program; Testing; Therapeutic; Time; Variant; Vial device; Viral; Viral Drug Resistance; Virus; Water; base; carboxylate; drug candidate; flu; improved; influenza epidemic; influenzavirus; luciferin; novel; pandemic disease; pandemic influenza; public health relevance; scale up; zanamivir

DESCRIPTION (provided by applicant): The primary goal of the Phase I SBIR projects is to determine the feasibility of a novel neuraminidase assay to be used as an influenza neuraminidase inhibition and drug susceptibility assay. The proposed assay is a biochemiluminescence assay that uses a novel substrate and permits real time determination of neuraminidase activity, which essentially requires only one preparation step - the addition of sample to a master detection mix that contains all necessary reagents for the assay. The proposed efforts include 1) improving substrate synthesis, 2) evaluating the feasibility as a NI and drug susceptibility assay, and 3) determining whether the assay has sufficient sensitivity so that clinical specimens can be directly used for the assay. Two neuraminidase inhibitors - zanamivir and particularly oseltamivir - are the current mainstay of pharmacological intervention during an influenza epidemic and, if happened, a pandemic. Emergence of drug resistant influenza virus variants heightens the concerns about widespread use of these drugs. It is imperative to monitor the emergence of antiviral drug resistant variants. Both cellular and biochemical based assays have been used to determine viral susceptibility to neuraminidase inhibitors. Cellular assays suffer from a number of drawbacks, including selection of mutations that compensate for neuraminidase inhibitor resistance during cell culture, long assay time, and tedious procedures, which make them less suitable for large scale surveillance programs. In contrast, the biochemical assays directly measure the inhibition of viral neuraminidase activities, thus circumventing many of the problems associated with the cellular assays. However, current biochemical assays are still complicated, take hours to complete, and have significant assay variability. More importantly, they lack sufficient sensitivity to allow drug susceptibility determination directly using clinical specimens (e.g., nasal wash), a much preferred sample type for large scale surveillance programs. Successful development of the proposed assay will allow the use of clinical specimens for drug susceptibility testing - a significant improvement over the currently used assays, which have to use virus isolates that are obtained via the lengthy and cumbersome culture methods. PUBLIC HEALTH RELEVANCE: Two neuraminidase inhibitors - zanamivir and oseltamivir - are the current mainstay of pharmacological intervention during an influenza epidemic or pandemic. Given the history of emergence of drug resistant influenza virus variants, it is imperative to monitor the emergence of antiviral drug resistant variants, which requires a robust neuraminidase inhibition (NI) assay. The proposed efforts are to determine the feasibility of a novel neuraminidase activity assay as an influenza NI assay. Specifically, we will determine if the assay is 1) rapid (5 min preparation time plus 30 min incubation); 2) simple (essentially one step operation); 3) sensitive (equivalent to or better than 25 cycles of real time PCR assay) so that clinical specimens can be directly used for NI assay; 4) reproducible (CV<15%); and 5) suitable for a 96-well format for large-scale sample detection.

描述（由申请人提供）：I阶段SBIR项目的主要目标是确定新型神经氨酸酶测定法的可行性，该测定法被用作流感神经氨酸酶抑制和药物敏感性测定。所提出的测定是一种生物化学测定法，该测定法使用新型底物，并允许对神经氨酸酶活性的实时确定，这基本上只需要一个制备步骤 - 将样品添加到包含所有必要试剂的主检测混合物中。拟议的努力包括1）改善底物合成，2）评估可行性为NI和药物敏感性测定法，3）确定测定法是否具有足够的敏感性，以便可以直接使用临床样本进行测定。两种神经氨酸酶抑制剂 - Zanamivir，尤其是Oseltamivir-是流感流行期间药理干预的当前中流，如果发生，则是大流行病。耐药性流感病毒变体的出现增强了人们对这些药物广泛使用的关注。必须监测抗病毒抗药性变体的出现。基于细胞和生化的测定法已用于确定对神经氨酸酶抑制剂的病毒敏感性。细胞测定法患有许多缺点，包括选择在细胞培养过程中补偿神经氨酸酶抑制剂耐药性的突变，较长的测定时间和繁琐的程序，这使得它们不太适合大规模监视程序。相反，生化测定直接测量了病毒神经氨酸酶活性的抑制，从而避免了与细胞测定相关的许多问题。但是，当前的生化测定仍然很复杂，需要花费数小时才能完成，并具有明显的变异性。更重要的是，它们缺乏足够的敏感性，无法使用临床标本（例如鼻腔清洗）直接确定药物敏感性，这是一种大规模监视程序的最喜欢的样品类型。拟议测定法的成功开发将允许使用临床标本进行药物敏感性测试 - 比当前使用的测定法，必须使用长期且繁琐的培养方法获得的病毒分离株。公共卫生相关性：两个神经氨酸酶抑制剂-Zanamivir和Oseltamivir-是流感流行或大流行期间药理干预的当前主要。鉴于耐药性流感病毒变体出现的历史，必须监测抗病毒药抗药性变异体的出现，这需要强大的神经氨酸酶抑制（NI）分析。拟议的努力是确定新型神经氨酸酶活性测定作为流感NI分析的可行性。具体而言，我们将确定测定是否为1）快速（5分钟准备时间加30分钟孵育）； 2）简单（本质上是一步操作）； 3）敏感（相当于或优于25个实时PCR分析周期），以便可以将临床标本直接用于NI分析； 4）可重现（CV <15％）； 5）适用于96孔格式用于大规模样品检测。