In this section

Dr Redhead, Intervet UK Ltd

Development of replacement in vitro assays for the quantification of clostridial vaccine antigens

Aims:

Clostridial bacteria produce toxins which cause a range of diseases in animals. Vaccines against these diseases are often developed from the toxin itself. The toxins can be treated to destroy their toxic properties, leaving a harmless form (a toxoid), which when used in a vaccine, is able to stimulate production of antibodies which will recognise the original clostridial toxin, thus protecting the vaccinated animal from disease.

During the manufacture of the vaccines the amounts of toxins and toxoids must be measured as a regulatory requirement, and to ensure consistent quality of the vaccines. Currently this involves the use of animals. However, there are in vitro approaches that could be exploited to measure these materials, thereby reducing and replacing the use of animals in toxin testing. The aim of this research is to develop in vitro assays to quantify the toxins and toxoids, to replace the current in vivo tests.

Methods

A wide range of cell lines has been developed, some of which are sensitive to certain clostridial toxins and display a detectable reaction to the toxin. These will be suitable cell lines to develop assays for some of the clostridial toxins and toxoids used in vaccine production. The cell lines will be investigated as methods for measuring the amount of toxin or toxoid present in a preparation, the degree of toxicity of specific clostridial toxins and the antigenicity (the ability to elicit an antibody response) of their respective toxoids.

The company which will carry out this research (Intervet) has also created many cell lines which produce antibodies specific for several different clostridial toxins. Antibodies can be developed which are specific for each clostridial toxin and toxoid. These antibodies can then be used in assays for toxins for which no suitable toxin-sensitive cell lines are available. This type of assay exploits the ability of specific antibodies to bind to the toxins and toxoids they were raised against. By measuring the amount of the antibody which binds to the toxin or toxoid, it is possible to estimate the amount of toxin or toxoid that is present. However, the quantity of a toxin or toxoid measured in an assay does not necessarily directly relate to its biological activity, i.e. toxicity or antigenicity.

The in vitro assays, using cell lines and antibodies, will be compared with in vivo tests, which have to be performed as part of the routine testing required by the regulatory authorities, and checked for correlation.

Implications for the 3Rs

Suitable in vitro assays could completely replace the use of animals in testing. As a veterinary vaccine manufacturer, Intervet produces a wide range of clostridial toxins and toxoids. To meet regulatory requirements, each batch of these materials is subjected testing. The replacement of these tests with in vitro assays would significantly reduce animal usage in vaccine production. In vivo tests are currently carried out as part of the normal vaccine manufacturing processes. This means that the necessary validations and correlations of the in vitro assays with in vivo tests can be done with minimal animal usage.