开发体外效力检测方法以替代肠道病毒71型灭活疫苗（人二倍体细胞基质/ Vero细胞基质）的体内试验
Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human Diploid Cell-Based/Vero Cell-Based).
作者信息
Zhang Xuanxuan, Yi Li, Yu Dan, Li Jun, Li Xintian, Wu Xing, Gao Fan, He Qian, Wang Wenhui, Wang Kaiwen, Wang Zejun, Liu Zhengling, Li Yadong, Zhao Yong, Li Huiyi, Ma Xiao, Zheng Qingbing, Xu Longfa, Cheng Tong, Zhu Rui, Guo Jing, Li Jing, Mao Qunying, Liang Zhenglun
机构信息
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, State Key Laboratory of Drug Regulatory Science, Research Units of Innovative Vaccine Quality Evaluation and Standardization, Chinese Academy of Medical Sciences, National Institutes for Food and Drug Control, Beijing 102629, China.
Institute of Medical Biology, Chinese Academy of Medical Sciences, Kunming 650118, China.
出版信息
Vaccines (Basel). 2025 Apr 13;13(4):404. doi: 10.3390/vaccines13040404.
BACKGROUND
The three commercial Enterovirus 71 (EV71) inactivated vaccines which have effectively controlled the EV71 pandemic currently rely on inherent variable in vivo potency methods for batch release. To align with 3R (Replacement, Reduction, Refinement) principles and enhance quality control, this study referred to WHO guidelines and the European Pharmacopoeia to develop in vitro relative potency (IVRP) methods.
METHODS
Working standards tracing to phase 3 clinical vaccines were established. Manufacture-specific IVRP methods were developed and validated per ICH Q14/Q2(R2), utilizing conformational epitope-targeting neutralizing monoclonal antibodies (MAbs). One of the MAbs (CT11F9) recognition sites was clarified with Cryo-EM. Subsequently, the performance of IVRP was assessed using varied concentrations and heat-treated vaccines. The correlation between IVRP and in vivo methods was analyzed, followed by setting IVRP specifications.
RESULTS
The manufacturer-specific working standard exhibited ED50 values comparable to those of related phase 3 clinical vaccines. All IVRP methods achieved a relative bias/precision/total error ≤ 15%. The IVRP methods correlated with in vivo methods ( < 0.05, r > 0.9) can discriminate EV71 antigen concentrations ( < 0.01, r > 0.99) and indicate the stability of the vaccines. Cryo-EM was adopted to identify the epitopes recognized by CT11F9, revealing that this neutralizing antibody recognizes a conformational epitope spanning VP1-3 of the same protomer. Using 31-47 batches of commercial vaccines, IVRP specifications were proposed as 0.56-1.35, 0.58-1.40, and 0.54-1.50.
CONCLUSIONS
Based on conformational epitope-targeting neutralizing MAbs, manufacturer-specific IVRP methods, which were sensitive to process variations and correlated with in vivo results, have been established. IVRP methods provide a reliable, animal-free alternative for EV71 vaccine batch release.
背景
目前三种有效控制肠道病毒71型（EV71）大流行的商业化EV71灭活疫苗在批签发时依赖固有的体内效价可变方法。为了符合3R（替代、减少、优化）原则并加强质量控制，本研究参考世界卫生组织指南和欧洲药典制定体外相对效价（IVRP）方法。
方法
建立了追溯至3期临床疫苗的工作标准品。根据国际人用药品注册技术协调会Q14/Q2（R2），利用靶向构象表位的中和单克隆抗体（MAb）开发并验证了各生产厂家特有的IVRP方法。通过冷冻电镜（Cryo-EM）阐明了其中一种单克隆抗体（CT11F9）的识别位点。随后，使用不同浓度和经过热处理疫苗评估IVRP的性能。分析IVRP与体内方法之间的相关性，接着设定IVRP规范。
结果
各生产厂家特有的工作标准品的半数有效剂量（ED50）值与相关3期临床疫苗相当。所有IVRP方法的相对偏差/精密度/总误差均≤15%。与体内方法相关（<0.05，r>0.9）的IVRP方法能够区分EV71抗原浓度（<0.01，r>0.99）并表明疫苗的稳定性。采用冷冻电镜鉴定CT11F9识别的表位，结果显示该中和抗体识别同一原体VP1-3上的一个构象表位。利用31-47批商业化疫苗，提出IVRP规范为0.56-1.35、0.58-1.40和0.54-1.50。
结论
基于靶向构象表位的中和单克隆抗体，已建立了各生产厂家特有的IVRP方法，该方法对工艺变化敏感且与体内结果相关。IVRP方法为EV71疫苗批签发提供了一种可靠的、无需动物的替代方法。
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