Replacement of hamsters with physicochemical analytical methods for Leptospira vaccine batch potency testing

Vaccination has been widely used to control leptospirosis in animals and consequently incidence in humans. Each vaccine batch must be tested for potency before market release. Hamsters are particularly sensitive to leptospirosis and are used as a model species to test the potency of vaccine batches. Typically five vaccinated and five unvaccinated hamsters are challenged with virulent Leptospira bacteria. Four of the vaccinates must survive and four unvaccinates must show signs of leptospirosis or die. The effects on the infected hamsters are severe (usually death), the test is time consuming (minimum duration 40 days) and expensive. Testing of the bivalent canine vaccine (L canicola and L icterohaemorrhagiae) is considered the most severe. The present study aims to replace the hamster model with quantitative mass spectrometry based assays for specific immunogenic vaccine components. Previous attempts by industry to define the components for batch testing have used monoclonal antibodies against protein and LPS immunogens but this approach is very specific, the analytes poorly defined and such antibodies are limited by their availability. The present study will use mass spectrometry based methods to quantitate immunogens to provide a transferable generic approach. Immunogenic components with be extracted from virulent and avirulent L canicola, and a commercial vaccine preparation, and fractionated by a range of techniques including size exclusion and ion exchange chromatographies. The active components will be detected using ELISA with immune serum from vaccinated hamsters, further purified as necessary and identified by mass spectrometry, principally MALDI-ToF/ToF. Protein immunogens will be quantitated using 13C and 15N labelled synthetic tryptic peptide and HPLC tandem mass spectrometry. LPS will be separated by gel chromatography and quantitated by MALDI-ToF. This will enable selected active components to be compared between batches of vaccines to ensure consistency of key immunogens. For simplicity, the project will focus on a single strain, such as L canicola, with the intention of providing proof of principle for this generic approach. Dissemination of information will be a critical success factor for the uptake and implementation of any replacement test by vaccine manufactures. The Veterinary Laboratories Agency has unique expertise in Leptospira vaccine batch testing for industry and consequently well placed with key contacts. Finally, this generic approach to vaccine batch testing using mass spectrometry based methods could potentially provide an alternative to the batch analysis of many other vaccine products currently tested using laboratory animals.