2015-10-24-世界卫生组织-WHO_BS_2015.2267_Report_on_the_WHO_collaborative_study_to_establish_the_1st_IS_for_anti-EV71_serum_Human_39页_847kb

报告摘要

Summary of the WHO Collaborative Study to Establish the 1st International Standard for Anti-EV71 Serum (Human)

Core Content

This document outlines the results of a World Health Organization (WHO) collaborative study aimed at establishing the 1st International Standard (IS) for anti-human enterovirus A71 (EV71) serum (Human). The study was conducted to standardize virus neutralization methods used to measure antibody levels against EV71 in human sera, which is critical for evaluating vaccine immunogenicity and assessing seroprevalence in human populations.

Main Objectives

To characterize two candidate serum samples (14/138 and 14/140) as potential International Standards for anti-EV71 serum.
To evaluate a third serum sample (13/238) as a WHO Reference Reagent for use as an assay control.
To ensure that the methods used for measuring neutralizing antibodies are accurate, sensitive, and reproducible across laboratories.

Key Information

Candidate Materials

Sample 14/138 and 14/140 (high titre): Pooled from 5 plasmas of healthy Chinese individuals with high EV71 neutralizing antibody (NTAb) titres and negative for CA16 antibodies.
Sample 13/238 (low titre): Pooled from 5 plasmas with low EV71 NTAb titres and negative for CA16 antibodies.
Sample 14/138 was recommended as the 1st IS with an assigned unitage of 1,000 IU/vial.
Sample 14/140 was proposed as a potential replacement for 14/138 with an assigned unitage of 1,090 IU/vial.
Sample 13/238 was proposed as a WHO Reference Reagent with an assigned value of 300 IU/vial.

Study Design

17 laboratories from the UK, USA, China, Taiwan, Japan, and Malaysia participated.
The study included national control laboratories, vaccine manufacturers, and public health laboratories.
Participants used their in-house virus neutralization methods and a common challenge virus (EV71 C4 523 strain) provided by NIBSC.
Laboratories 14 and 17 also used the EV71 B4 C7/Osaka/1997 strain as a second challenge virus.

Sample Preparation and Testing

Plasma samples were collected from healthy individuals in China and tested for EV71 NTAb titres.
Defibrination and pooling were performed to produce the candidate sera.
Freeze-drying was carried out using a 2-day cycle, with 3ml ampoules filled with 0.5ml of material.
Post-filling testing showed no significant loss of activity in the freeze-dried samples.
Stability studies were conducted at various temperatures and showed that the samples retained activity when stored at -20°C and below for up to 6 months.

Assay Methodology

All participants used a constant virus varying serum method with RD cells.
The challenge dose was recommended to be between 1.0–3.0 log₁₀ TCID₅₀.
Intra-assay variability was assessed using coded duplicates, showing a good level of precision (97% of relative potencies within [0.5, 2.0]).
Inter-assay variability was assessed using geometric coefficients of variation (GCV), ranging from 0% to 396%, with no further exclusions made for the analysis.

Statistical Analysis

The pooled between-laboratory GCV for clinical samples (T, W, X, Y) was 64% for end point titres and reduced to 41% for potencies relative to 14/138.
For potencies relative to 14/140, the pooled GCV was 35%.
Vero vs. RD cell methods showed no significant difference in results, with RD cells being the most commonly used.

Conclusion

The study successfully characterized and validated the 1st International Standard for anti-EV71 serum (Human), with sample 14/140 recommended as the standard due to its consistent performance and high antibody titre. The low titre sample (13/238) was proposed as a WHO Reference Reagent. The results demonstrate that the neutralization assays can be standardized and used globally for evaluating vaccine immunogenicity and seroprevalence. The use of a common challenge virus helped reduce variability, and the stability data support the long-term use of these materials.

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