Epsilon Open Archive

Abstract

The intracellular protozoan parasite Neospora caninum is an important cause of bovine abortion and congenital infection in many countries. Currently there is no effective control method available, and development of a vaccine has been suggested as a possible strategy to prevent the disease neosporosis. In this thesis an experimental recombinant subunit vaccine, consisting of the N. caninum antigen NcSRS2 and immunostimulating complexes (iscoms), was evaluated. The iscom is an adjuvant formulation into which antigens can be incorporated by hydrophobic interactions. Many recombinant proteins are hydrophilic, and special methods are needed for binding them to the iscoms and to achieve optimal adjuvant effects. A novel method to combine recombinant proteins with iscoms utilizing the strong interaction between biotin and streptavidin was evaluated. Recombinant NcSRS2 was expressed in Escherichia coli as two different fusion proteins. One of them was biotinylated in E. coli and was bound to streptavidin-coated iscom matrix (iscom particles without any antigen). The other fusion protein contained recombinant streptavidin and was bound to biotinylated iscom matrix. These two strategies both resulted in efficient binding to the iscom matrix. Thus, this method might offer a convenient and effective alternative for association of other hydrophilic recombinant antigens with iscoms for evaluation in immunisation experiments. Both the immunogenicity and the protective effect of the NcSRS2-iscoms were investigated by immunisation and challenge infection of mice. A real-time PCR assay was developed, and used for quantification of parasite DNA in the brain and blood of infected mice. The NcSRS2-iscoms induced production of antibodies that recognised NcSRS2 of parasite origin, suggesting that the conformation of the recombinant protein was similar to that of the native antigen. Antigen-specific cellular responses were also induced as demonstrated by in vitro proliferation and cytokine production. After challenge infection, the immunised mice had significantly lower levels of N. caninum DNA in their brains and blood than did non-immunised control mice. These mice were also less affected by the disease, as judged by clinical symptoms and changes in body weight. These results, together with results from other studies, indicate that recombinant NcSRS2 might be considered as a potential candidate antigen for a future subunit vaccine against N. caninum infection in cattle.