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.

In vitro determination of specific toxicity in tetanus vaccines.

Tetanus vaccine is prepared from detoxified tetanus neurotoxin. To ensure the absence of residual toxin activity or to exclude the reversion to toxicity reliable control testing is based on in vivo methods, because no in vitro assay provides the required specificity and sensitivity. Tetanus neurotoxin is a 150 kDa protein produced by Clostridium tetani. The 50 kDa light chain of this neurotoxin belongs to the family of zinc metalloproteases. It cleaves synaptobrevin, a small synaptic vesicle protein, which is involved in neuroexocytosis, at the single Q76-F77 peptide bond. To develop a sensitive in vitro assay capable of quantifying the proteolytic activity of this toxin, we used as substrate a recombinant fragment of synaptobrevin2 (1-97). For detecting the cleavage products a peptide antibody raised against the N-terminal cleavage site was used. In Western Blot analysis only the cleaved substrate was detected while the uncleaved substrate showed no signal. In different approaches, recombinant synaptobrevin was either (i) bound to a microtitre plate, reduced toxin was added and the N-terminal cleavage product was detected by a specific antibody or (ii) the cleavage was performed in test tubes, the samples were transferred to a microtitre plate and immobilised cleavage products were detected. When toxoid or crude toxin is used, non-specific cleavage of synaptobrevin substrate occurs. Depending on the toxoid used different patterns of degradation of substrate are visible in Western Blots. Different protease inhibitors and reaction conditions seem to have an effect on the inhibition of this non-specific cleavage.