The rapid fluorescent focus inhibition test is a suitable method for batch potency testing of inactivated rabies vaccines.

The European Pharmacopoeia proposes two methods for potency determination of inactivated rabies vaccines for veterinary use: The first one is a classical mouse challenge test, which is imprecise, time-consuming, and causes severe distress to the test animals. Alternatively, the potency may be determined serologically by measuring the neutralizing antibody titers induced after vaccination of mice by using a rapid fluorescent focus inhibition test (RFFIT). Although this method is faster and less painful for the animals, it is not widely used yet, and only little data exist concerning the comparability of both methods. We have therefore performed a comparative study, in which we demonstrated a good correlation between the challenge test results and the mean titers determined by RFFIT. Furthermore, all vaccine batches failing the challenge test were also recognized as insufficient in the serological assay. This publication further describes the influence of different vaccine administration routes on the resulting antibody titers, and it proposes various modifications to the serological assay protocol which could improve its overall practicability. Finally, we recommend that the serological assay be used for the potency testing of inactivated rabies vaccines.

Residual enzymatic activity of the tetanus toxin light chain present in tetanus toxoid batches used for vaccine production.

The light chain of tetanus neurotoxin (TeNT) is a zinc-dependent metalloprotease which specifically cleaves the synaptic vesicle protein synaptobrevin. This crucial mechanism of tetanus toxicity leads to a blockade of inhibitory neurotransmitter release. We recently reported the development of a highly sensitive endopeptidase assay for the specific in vitro detection of active TeNT based on this proteolytic feature. Using this method, we could show that formaldehyde-inactivated TeNT preparations (toxoids), which are used for the production of tetanus vaccines, contain a high residual synaptobrevin-cleaving activity. Such an activity was detected in numerous tetanus toxoid batches obtained from several vaccine manufacturers which did not display any in vivo toxicity in the obligatory animal tests. The enzymatic activity could be attributed to the presence of free TeNT light chains whose function had not been restrained by the formaldehyde treatment, but which lack the functional heavy chain necessary for entering neurons in vivo. To our knowledge, this is the first report describing a residual proteolytic activity in tetanus toxoids.

An in vitro assay for detection of tetanus neurotoxin activity: using antibodies for recognizing the proteolytically generated cleavage product.

Tetanus neurotoxin (TeNT(1)) is a bacterial protease which specifically cleaves the vesicle protein synaptobrevin-2 (vesicle associated membrane protein-2, VAMP-2). This proteolytic feature of the toxin has been used to develop a sensitive endopeptidase assay for the detection of TeNT activity as an alternative to the in vivo assay for TeNT toxicity. Recombinant synaptobrevin-2 (rSyb2) is immobilized onto a microtiter plate, and the cleavage of immobilized rSyb2 by TeNT is detected with a polyclonal antibody directed against the newly generated C-terminus of the cleavage product. This antibody is shown to be a highly specific tool for detecting rSyb2 proteolysis by TeNT. The method reaches a detection limit of less than 1pg TeNT/ml. To our knowledge, this is the most sensitive in vitro assay for the detection of TeNT activity, and it is easy to perform. Besides, the assay can also detect the activity of botulinum neurotoxin type B (BoNT/B). The method can be applied to examine the toxicity of TeNT or BoNT/B preparations as well as the influence of chemicals on TeNT and BoNT/B activity. In the future, the assay may also serve as a basis for the replacement of the in vivo safety control of tetanus vaccines.