DataSheet_1_H3N2 influenza hemagglutination inhibition method qualification with data driven statistical methods for human clinical trials.docx

IntroductionHemagglutination inhibition (HAI) antibody titers to seasonal influenza strains are important surrogates for vaccine-elicited protection. However, HAI assays can be variable across labs, with low sensitivity across diverse viruses due to lack of standardization. Performing qualification of these assays on a strain specific level enables the precise and accurate quantification of HAI titers. Influenza A (H3N2) continues to be a predominant circulating subtype in most countries in Europe and North America since 1968 and is thus a focus of influenza vaccine research.
MethodsAs a part of the National Institutes of Health (NIH)-funded Collaborative Influenza Vaccine Innovation Centers (CIVICs) program, we report on the identification of a robust assay design, rigorous statistical analysis, and complete qualification of an HAI assay using A/Texas/71/2017 as a representative H3N2 strain and guinea pig red blood cells and neuraminidase (NA) inhibitor oseltamivir to prevent NA-mediated agglutination.
ResultsThis qualified HAI assay is precise (calculated by the geometric coefficient of variation (GCV)) for intermediate precision and intra-operator variability, accurate calculated by relative error, perfectly linear (slope of -1, R-Square 1), robust (<25% GCV) and depicts high specificity and sensitivity. This HAI method was successfully qualified for another H3N2 influenza strain A/Singapore/INFIMH-16-0019/2016, meeting all pre-specified acceptance criteria.
DiscussionThese results demonstrate that HAI qualification and data generation for new influenza strains can be achieved efficiently with minimal extra testing and development. We report on a qualified and adaptable influenza serology method and analysis strategy to measure quantifiable HAI titers to define correlates of vaccine mediated protection in human clinical trials.

引言：针对季节性流感毒株的血凝抑制（Hemagglutination Inhibition, HAI）抗体滴度是疫苗诱导保护作用的重要替代指标。然而，HAI试验在不同实验室间结果存在差异，且由于缺乏标准化流程，对多种病毒的检测灵敏度偏低。针对毒株特异性层面开展此类试验的确证工作，可实现HAI滴度的精准定量。自1968年以来，甲型流感病毒（H3N2）始终是欧洲与北美多数国家的主要流行亚型，因此成为流感疫苗研发的重点方向。
方法：作为美国国立卫生研究院（National Institutes of Health, NIH）资助的合作流感疫苗创新中心（Collaborative Influenza Vaccine Innovation Centers, CIVICs）项目的组成部分，本研究报道了一项稳定试验设计的确立、严格的统计分析，以及以A/Texas/71/2017作为代表性H3N2毒株、豚鼠红细胞以及神经氨酸酶（neuraminidase, NA）抑制剂奥司他韦（用于阻断NA介导的凝集反应）来完成HAI试验的确证工作。
结果：该确证后的HAI试验在中间精密度与操作者内变异度方面均表现出优异的精密度（以几何变异系数（geometric coefficient of variation, GCV）计算），以相对误差衡量的准确度符合要求，线性关系极佳（斜率为-1，决定系数R²=1），稳定性良好（GCV<25%），且具备较高的特异性与灵敏度。该HAI方法还成功完成了另一株H3N2流感毒株A/Singapore/INFIMH-16-0019/2016的试验确证，满足所有预先设定的接受标准。
讨论：本研究结果表明，针对新型流感毒株开展HAI试验确证与数据生成工作，可通过最少的额外试验与开发步骤高效完成。本研究报道了一种经过确证且可适配的流感血清学试验方法与分析策略，用于定量检测HAI滴度，以明确人类临床试验中疫苗介导保护作用的相关指标。