Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA - Results of an international collaborative study.

Three different ELISAs quantifying rabies glycoprotein were evaluated as in vitro alternatives to the National Institutes of Health (NIH) in vivo potency test for batch release of human rabies vaccines. The evaluation was carried out as an international collaborative study supported by the European Partnership for Alternatives to Animal Testing (EPAA). This pre-validation study, the results of which are presented in this paper, compared three different ELISA designs, assessing their within- and between-laboratory precision. One of the ELISA designs was proposed to the European Directorate for the Quality of Medicines & HealthCare (EDQM) and accepted for an international collaborative study under the umbrella of the Biological Standardisation Programme.

Development and validation of a quantitative competitive ELISA for potency testing of equine anti rabies sera with other potential use.

In case of a bite by a rabies infected animal, the World Health Organisation recommends a prophylactic treatment including the administration of Human Rabies Immunoglobulins (HRIGs) or highly purified F(ab')2 fragments produced from Equine Rabies Immunoglobulin (F(ab')2 - ERIGs). According to international regulation, quality control of F(ab')2 - ERIGs lots requires potency testing by the in vivo Mouse Neutralisation Test (MNT) prior marketing. However, the strategy of the 3Rs (Reduce, Refine, Replace) for animal testing required by the European Directive encourages the replacement of the in vivo potency test by an in vitro assay. In this context, a competitive ELISA method (c-ELISA) has been developed by the Agence Nationale de Sécurité du Médicament et des Produits de Santé where F(ab')2 - ERIGs are in competition with a monoclonal antibody recognizing the trimeric native form of the rabies glycoprotein. After a full validation study, the c-ELISA has been applied to commercial batches of F(ab')2 - ERIGs. A correlation study with the MNT demonstrated a similarity between the two methods (r=0.751). Moreover, the c-ELISA method which does not need any species specific reagent has been applied to HRIGs potency testing as an alternative method to Rapid Fluorescent Focus Inhibition Test (RFFIT), thus avoiding the handling of live rabies virus in BSL3 containment. In conclusion, the c-ELISA has shown its potential to replace MNT and possibly RFFIT for the quantification of rabies immunoglobulin. After optimisation it may be used for the quantification of rabies immunoglobulin in any animal species, notably for rabies immunogenicity assay in mice.

A relevant in vitro ELISA test in alternative to the in vivo NIH test for human rabies vaccine batch release.

To assess the quality of vaccine batches before release, international regulation requires the control of potency of each lot of human rabies vaccines by the in vivo NIH challenge test. Meanwhile, the 3Rs strategy for animal testing encourages the replacement of the in vivo potency test by an in vitro assay. Consequently, since more than 10 years, an ELISA method has been implemented by ANSM in parallel to the NIH test for rabies vaccines lots. It consists in the evaluation of the glycoprotein content using a monoclonal antibody recognizing the trimeric native form of the glycoprotein. This ELISA method is able 1) to monitor the consistency of production with a similar profile than the NIH; 2) to detect a low quantity of glycoprotein in vaccines and 3) to agree with the manufacturer's NIH results by declaring a non compliant batch. This ELISA which characterizes the immunogenic form of the glycoprotein formulated in vaccines seems to be relevant to replace the NIH test and is a promising candidate to be standardized by a collaborative study.

Would an in vitro ELISA test be a suitable alternative potency method to the in vivo immunogenicity assay commonly used in the context of international Hepatitis A vaccines batch release?

Since many years Afssaps applies the 3R's strategy (replacement, reduction and refinement) for the use of laboratory animal testing in the framework of vaccine batch release. In this context, for Hepatitis A vaccines, a study was carried out to assess the feasibility of replacing the in vivo "Gold Standard" potency assay by the Afssaps' validated in-house antigen content in vitro assay on routine testing. The use of a panel of potent vaccine batches and reduced-potency samples by heating demonstrated a correlation between the two methods. This encourages Afssaps to progressively switch from in vivo to in vitro assay in the framework of Hepatitis A vaccines batch release.

The assessment of OPV vaccines by the monkey neurovirulence test: why and how to qualify the experts in histology of central nervous system.

Prior to batch release of oral poliovirus vaccines (OPV) for marketing purpose, the World Health Organisation (WHO) and European pharmacopoeia require a monkey neurovirulence test to be performed both by the manufacturer and the relevant National Control Laboratory (NCL) to assess vaccine safety as regards neurovirulence. Due to the subjectivity of histological examination and of neural lesions scoring, the French NCL has set up a proficiency testing procedure to qualify a new expert.