In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

Summary

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Abstract

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.

The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Introduction

Since more than 50 years, the NIH test¹ is used as a gold standard method to evaluate the rabies vaccine potency before the batch release. This test consists of an intraperitoneal immunization of groups of mice with the vaccine to be tested followed by an intra-cerebral (IC) challenge 14 days later with the Challenge Virus Standard (CVS) strain of rabies virus (RABV). The potency is evaluated from the proportion of mice surviving the IC challenge. Although WHO² and European Pharmacopeia³ still require the NIH test for assessing the vaccine potency, this test suffers several hurdles: results are hi....

Protocol

1. Security precautions

NOTE: This method is applicable to both live RABV and inactivated vaccine.

1. Use good Laboratory Practice and Safety procedures.
2. Wear adequate Personal Protection Equipment (PPE) including disposable coat, gloves, mask, glasses, etc.
3. When the live virus is titrated, use a class II biological safety cabinet.
   1. Consider any material in contact with the samples (reagents, washing solutions, etc.) as infectious material.
   2. Treat the contaminated material by immersing in the bleach solution (2,5% of sodium hypochlorite) for 30 min for decontamination.

Results

In the following example the reference vaccine Lot 09, consisting of purified inactivated rabies virus particles (PV vaccine strain), is used. The glycoprotein trimers (10 µg/mL) content in this has been established after the determination of the total amount of viral proteins (BCA test) and evaluation of the percentage of the glycoprotein by SDS-polyacrylamide gel electrophoresis. Alternatively, a calibrated reference vaccine, e.g., the WHO 6^th International Standard (IS) for .......

Discussion

The epitope recognized by the mAb-D1 is located in the antigenic site III of the RABV glycoprotein which is not only immuno-dominant for the induction of VNAbs but also involved in neurovirulence, pathogenicity^40,41 and receptor recognition⁴². There is another important antigenic site along the glycoprotein, site II⁴³, against which several MAbs have been isolated such as mAb-WI-1112³⁵. These.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Class II Biological Safety Cabinet	ThermoFisher Scientific	10445753	if titrating live virus
Clear Flat-Bottom Immuno Nonsterile 96-Well Plates, 400 µL, MAXISORP	ThermoFisher Scientific	439454	good for binding to the loaded antibody
Equip Labo Polypropylene Laboratory Fume Hood	ThermoFisher Scientific	12576606	for the preparation of sulfuric acid
Immunology Plate Strong Adsorption MAXISORP Flat Bottom Well F96	Dutscher	55303	good for binding to the loaded antibody
Microplate Sealing Tape(100 sheets)	ThermoFisher Scientific	15036
Microplate  single mode reader Sunrise	TECAN
Microplate shaker-incubator	Dutscher	441504
Microplate washer Wellwash	ThermoFisher Scientific	5165000
Multichannel pipette (30-300 µL) 12 channels	ThermoFisher Scientific	4661180N
Single Channel pipettes (Kit 2 : Finnpipettes F2 0.2-2 μL micro, 2-20 μL, 20-200 μL & 100-1000 μL)	ThermoFisher Scientific	4700880