Europ. collab. trial – an alternat. method to animal exp. for the safety testing of tetanus vaccines

Vaccines for human and veterinary use must be efficacious and safe. The European Pharmacopeia stipulates how vaccines have to be experimentally tested, i.e. with laboratory methods and/or animal experiments. For many tests, animal experiments are still compulsory – safety testing (toxicity test) of tetanus vaccines included. For these vaccines, an innocuous toxoid is produced by chemical inactivation from the toxin of the bacterium Clostridium tetani. This toxoid is then administered as vaccine provoking the immune response. It is important to ensure that the vaccine itself does not contain any active tetanus neurotoxin. The currently prescribed toxicity test is performed in guinea pigs. If the toxoids are not sufficiently inactivated, the experimental animals develop tetanus symptoms.

Researchers at the Paul-Ehrlich-Institut have developed a so-called „BINACLE“ (binding and cleavage) assay that – in a combined test system – examines not one but two characteristic properties of the toxin which are also responsible for its toxic effect: the toxin’s ability to bind to specific receptor molecules on nerve cells and the ability to cleave a substrate protein inside the nerve cell. The research team was awarded the German Animal Protection Research Prize in 2010 for the development of this animal-free test system.

In the meantime, the working group was able to show that the method can be used for various licensed tetanus vaccines with the detection limit for the neurotoxin being equivalent to the estimated detection limit of the animal experiment [1]. Furthermore, the transferability of the test system was demonstrated in an international study using the BINACLE method in different laboratories [2]. However, prior to being incorporated in the European Pharmacopeia and thus become binding, the method must be tested successfully in a European collaborative trial. To this end, various testing laboratories – OMCLs (Official Medicines Control Laboratories), national control authorities of different European and non-European countries and vaccine manufacturers – apply the new method to standardised samples and determine the reliability of the method. If the collaborative trial proves successful, too, an important hurdle would be taken on the way towards including a new animal-free test in the European Pharmacopoeia. The starting signal for performing this trial was given with the distribution of the test reagents to the study participants.

The collaborative trial is organised and financed by the EDQM. The project leaders at the Paul-Ehrlich-Institut’s Veterinary Division are Dr Heike Behrensdorf-Nicol and Dr Beate Krämer, who have played a vital part in the conceptualisation of the collaborative trial. In addition, they were responsible for designing a detailed test protocol, testing the samples in preparation of the study and providing specific materials for the trial.

The development of the method was supported by the Federal Ministry of Education and Research, the SET Foundation, the Animalfree Research Foundation and the Doerenkamp-Zbinden Foundation.

Publications of the working group on this subject:

Behrensdorf-Nicol HA, Bonifas U, Hanschmann KM, Krämer B, Weißer K (2013): Binding and cleavage (BINACLE) assay for the functional in vitro detection of tetanus toxin: Applicability as alternative method for the safety testing of tetanus toxoids during vaccine production.
Vaccine 31: 6247-6253.

Behrensdorf-Nicol A, Bonifas U, Isbrucker R, Ottiger H, Tierney R, Hanschmann KM, Weisser K, Krämer B (2014): Results of an international transferability study of the BINACLE (binding and cleavage) assay for in vitro detection of tetanus toxicity.
Biologicals 42: 199-204.

Behrensdorf-Nicol HA, Bonifas U, Kegel B, Silberbach K , Krämer B, Weisser K (2010): In vitro determination of tetanus toxicity by an endopeptidase assay linked to a ganglioside-binding step.
Toxicol In Vitro 24: 988-94.

Behrensdorf-Nicol HA, Kegel B, Bonifas U, Silberbach K, Klimek J, Weißer K, Krämer B (2008): Residual enzymatic activity of the tetanus toxin light chain present in tetanus toxoid batches used for vaccine production.
Vaccine 26: 3835-3841.

Kegel B, Behrensdorf-Nicol HA, Bonifas U, Silberbach K, Klimek J, Krämer B, Weißer K (2007): An in vitro assay for detection of tetanus neurotoxin activity: Using antibodies for recognizing the proteolytically generated cleavage product.
Toxicol in Vitro 21: 1641-1649.

The Paul-Ehrlich-Institut, the Federal Institute for Vaccines and Biomedicines, in Langen near Frankfurt/Main is a senior federal authority reporting to the Federal Ministry of Health (Bundesministerium für Gesundheit, BMG). It is responsible for the research, assessment, and marketing authorisation of biomedicines for human use and immunological veterinary medicinal products. Its remit also includes the authorisation of clinical trials and pharmacovigilance, i.e. recording and evaluation of potential adverse effects.

Other duties of the institute include official batch control, scientific advice and inspections. In-house experimental research in the field of biomedicines and life science form an indispensable basis for the manifold tasks performed at the institute.

The Paul-Ehrlich-Institut, with its roughly 800 members of staff, also has advisory functions nationally (federal government, federal states (Länder)), and internationally (World Health Organisation, European Medicines Agency, European Commission, Council of Europe etc.).

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