A novel electrophoretic immunoblot as antigen desorption and quantification method for alum-adjuvanted veterinary rabies vaccines.

Rabies vaccines for domestic animals are adjuvanted with aluminum salts. A particular challenge for in-vitro batch potency tests with these products is the fact that the antigens are firmly adsorbed to the aluminum salt matrix and thus are not easily available for antigen quantification. In the current manuscript we describe a versatile technique to quantify antigens in aluminum adsorbed vaccine formulations. A combined electrophoretic desorption and blotting method is presented that transfers the antigens to a nitrocellulose membrane followed by an immunoblot quantification of the transferred rabies antigens. For the immunoblot a rabies G-protein specific, monoclonal antibody is used that by itself has neutralizing activity. This ensures that only relevant antigens are quantified. By comparing end products with non-adjuvanted in-process material it can be demonstrated that the antigens are quantitatively desorbed from the adjuvant matrix. Resuts of the new antigen quantification method were compared with the outcome of the serological batch potency test as described in the European Pharmacopoeia. It is demonstrated that the new antigen quantification method reveals relevant differences between experimental vaccine batches formulated with increasing antigen loads. This proves the broad detection range of the method. In general, the results show that this highly versatile technique can serve as an important component of a comprehensive consistency test strategy and may be applied in a modified form to any alum-adjuvanted vaccine.

Vaccination against Feline Panleukopenia: implications from a field study in kittens.

BACKGROUND: Feline Panleukopenia (FPL) is a serious disease of cats that can be prevented by vaccination. Kittens are routinely vaccinated repeatedly during their first months of life. By this time maternally derived antibodies (MDA) can interfere with vaccination and inhibit the development of active immunity. The efficacy of primary vaccination under field conditions was questioned by frequent reports to the Paul-Ehrlich-Institut on outbreaks of FPL in vaccinated breeding catteries. We therefore initiated a field study to investigate the development of immunity in kittens during primary vaccination against FPL.64 kittens from 16 litters were vaccinated against FPL at the age of 8, 12 and 16 weeks using three commercial polyvalent vaccines. Blood samples were taken before each vaccination and at the age of 20 weeks. Sera were tested for antibodies against Feline Panleukopenia Virus (FPV) by hemagglutination inhibition test and serum neutralisation assay in two independent diagnostic laboratories. RESULTS: There was a good correlation between the results obtained in different laboratories and with different methods. Despite triple vaccination 36.7% of the kittens did not seroconvert. Even very low titres of MDA apparently inhibited the development of active immunity. The majority of kittens displayed significant titres of MDA at 8 and 12 weeks of age; in some animals MDA were still detected at 20 weeks of age. Interestingly, the vaccines tested differed significantly in their ability to overcome low levels of maternal immunity. CONCLUSIONS: In the given situation it is recommended to quantify antibodies against FPV in the serum of the queen or kittens before primary vaccination of kittens. The beginning of primary vaccination should be delayed until MDA titres have declined. Unprotected kittens that have been identified serologically should be revaccinated.