Entwicklung und Charakterisierung eines kontrollierten Freisetzungssystems für die in ovo-Vakzinierung beim Huhn

Despite vigorous vaccination strategies in the field, health problems continue to be of major economic importance for commercial farm animals, especially poultry. New generation vaccines such as recombinant plasmid DNA (pDNA) vaccines encoding for antigens of pathogens offer new opportunities for the induction of protective immunity in vaccinated animals. For commercial poultry vaccination, pDNA has not been practicable so far, because these vaccines have to be administered individually via parenteral routes. Recent studies have shown that encapsulation of pDNA in microspheres will protect the pDNA and even oral application may be possible. Depending on the configuration of the microspheres the release of the pDNA may be delayed or phasic. The possible use and safety of microspheres as a controlled release system for pDNA-vaccines for poultry has not been tested yet. The goal of this study was to develop a microsphere based controlled release system for pDNA vaccination of chickens. The specific focus was on the in ovo route for vaccine administration, because this route is successfully used in the field for mass vaccination. Different protocols for the production of microspheres an a variety of configurations were tested using the emulsification/solvent evaporation method. The following parameters were investigated: size of microspheres, being important for their take-up by macrophages; pDNA encapsulation rate; pDNA release; pDNA integrity. The in vitro take-up of microspheres by macrophages, their stimulation, and in vitro expression of the model antigen chloramphenicol aminotransferase (CAT) encoded by the enclosed pDNA was tested. Furthermore, the safety of the microsphere preparations was investigated after in ovo vaccination as well as after intramuscular and subcutaneous vaccination of post hatch chickens. A variety of poly(lactide-co-glycolide) formulations in combination with different solvents, surfactant factors, and pDNA stabilising factors were tested. The best pDNA encapsulation rate with microsphere size distribution between 5 - 10 µm was obtained with Lactel®BP-0100 and Lactel®50DG040 or their mixture with Resomer®RG 503H. These microspheres were prepared with dichloromethane and ethyl acetate, and were taken up by chicken macrophage cells, which successfully expressed CAT. Furthermore, it was demonstrated that these microspheres were non-toxic for chickens or embryos, which had received the microspheres after hatch or at 6 or 18 days of embryonation, respectively. Overall, in this study it was demonstrated that microspheres prepared with the described protocols were non-toxic for chickens and chicken macrophages and can be considered safe as a potential controlled release system for pDNA-vaccines in chickens. Future studies have to confirm the expression of the encapsulated pDNA encoded antigens and the development of protective immunity after in vivo administration.