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.

Bovine Neonatal Pancytopenia: is this alloimmune syndrome caused by vaccine-induced alloreactive antibodies?

Bovine Neonatal Pancytopenia (BNP) is a new emerging disease observed since 2007 in Germany and neighbouring countries. The syndrome affects newborn calves and is characterized by pancytopenia, severe bleeding and high lethality. So far, a causative role of infectious or toxic agents has been ruled out. Instead, the syndrome is induced after ingestion of colostrum, the first milk that supplies the calf with maternal antibodies. In analogy to similar diseases in humans it has therefore been postulated that BNP is caused by alloreactive, maternal antibodies. There is a striking association between BNP and a previous vaccination of the respective dams with a particular vaccine against Bovine Virus Diarrhoea (BVD). This association has led to a suspension of the marketing authorisation for the vaccine, by the European Commission. The current study investigates the role of this vaccine in the pathogenesis of BNP. By flow cytometry we were able to demonstrate that sera of BNP dams (dams that gave birth to a BNP calf) harbour alloreactive antibodies binding to surface antigens on bovine leukocytes. A significantly weaker alloreactivity was observed with sera of non-BNP dams that have been vaccinated with the same vaccine but delivered healthy calves. No binding was seen with non-BVD-vaccinated control cows and animals that were vaccinated with other inactivated BVD vaccines so far not associated with BNP. The binding is functionally relevant, because opsonization of bovine leukocytes with alloantibodies led to an elevated cytophagocytosis by bovine macrophages. To test whether the vaccine induces alloreactive antibodies two strategies were employed: Guinea pigs were vaccinated with a panel of commercially available BVD-vaccines. Only the incriminated vaccine induced antibodies binding surface antigens on bovine leukocytes. Additionally, two calves were repeatedly vaccinated with the suspected vaccine and the development of alloreactivity was monitored. In dependence of the number of booster immunizations the induction of alloreactive antibodies could be observed. Finally, by affinity purification we were able to directly demonstrate that BNP associated alloantibodies cross react with the bovine kidney cell line used for vaccine production. Together this provides strong evidence that this particular BVD vaccine has the potential to induce BNP associated alloantibodies.