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Updated: May 14, 2025
In Vitro ELISA Test to Evaluate Rabies Vaccine Potency
Published on: May 11, 2020
Development of In Vitro Potency Methods to Replace In Vivo Tests for Enterovirus 71 Inactivated Vaccine (Human
Xuanxuan Zhang1, Li Yi2, Dan Yu3
1NHC 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.
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 (p < 0.05, r > 0.9) can discriminate EV71 antigen concentrations (p < 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.
