Replacement in vitro assays for the quantification of clostridial vaccine antigens

Clostridia are responsible for a wide range of animal diseases, and clostridial vaccines are among the most common veterinary biologicals. Clostridial diseases are mediated by toxins and vaccines comprise toxoided forms of these toxins. During manufacture toxin production is measured and the amount of toxoid antigen quantified. These stages rely on animal testing. The toxins are measured by minimum lethal dose (MLD) and/or L+ tests in mice and the toxoids by total combining power (TCP) assays, in mice. The replacement of these tests with in vitro assays would significantly reduce animal usage in vaccine production. 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 to MLD, L+ and TCP testing. As part of our ongoing research, supported by the NC3Rs, we have been looking at the possibility of developing in vitro assays based on the use of cell lines to detect clostridial toxins and to quantify clostridial antigens. The intention is to develop in vitro assays to replace the current in vivo tests. Cell lines have been investigated as methods for measuring the toxicity of specific clostridial toxins and the antigenicity of their respective toxoids. The in vitro assays are compared with in vivo tests, which have to be performed as required by the regulatory authorities, and checked for correlation. One major advantage is that the performance of the in vivo tests, is part of the normal vaccine manufacturing processes. This means that the necessary validations and correlations with in vivo tests can be done with minimal animal usage. Cell lines have been identified that have suitable specific sensitivities for the clostridial toxins of interest. In the one case alternative cell line assays have been developed, validated and are currently in the latter stages of correlation studies. So proving the principles of this approach. However, within the time span of the current NC3Rs grant it will not be possible to develop, validate and correlate equivalent in vitro assays for all of the common clostridial vaccine antigens. An additional one year grant would make this goal achievable. The potential reward for replacing these tests is great. Intervet, as just one veterinary vaccine manufacturer, uses an average of 8500 mice per year performing these tests on clostridial components.

• 2011 Research review: Developing in vitro assays to replace animal tests for clostridial vaccine antigens
• Further Funding: NC3Rs Project Grant, Development of replacement in vitro assays for the quantification of clostridial vaccine antigens, March 2006, £111,901