Abstract
More than a million animals are used annually in the European Union for regulatory batch testing of medicinal products, predominantly for potency assessment. With respect to inactivated poultry vaccines, most potency tests are still performed in vivo due to the lack of in vitro alternatives. It is important to move
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away from in vivo testing because of ethical concerns, variability, long duration and high costs of these tests, risks for personnel working with infected animals, and legal requirements to use in vitro methods instead of in vivo methods whenever possible. The primary aim of this thesis was to set up in vitro cell-based assays to assess the immunostimulatory capacity of inactivated poultry vaccines and to determine their applicability for routine potency testing.
The studies presented in Chapters 2 and 3 of this thesis focus on the immunostimulatory properties of inactivated poultry vaccines by investigating their effect on chicken macrophages. The cell-based assays described involve the use of the macrophage-like cell line HD11. A phagocytosis assay with IgY-opsonized beads was used to investigate whether inactivated poultry vaccines against infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and egg-drop syndrome virus (EDSV) affect phagocytosis by macrophages. In addition, the use of nitric oxide production and cytokine gene expression are described to determine the immunostimulatory properties of an inactivated octavalent vaccine against IBV, NDV, EDSV and five strains of Avibacterium paragallinarum, the causative agents of infectious coryza characterized by an acute respiratory disease in chickens.
The study presented in Chapter 4 describes a thorough morphological, phenotypical and functional characterization of chicken bone marrow-derived dendritic cell cultures. In Chapter 5, bone marrow-derived dendritic cells were exposed to an inactivated poultry vaccine against IBV and NDV to identify biomarkers of vaccine-induced immune responses by proteomic analysis. Furthermore, the mechanisms by which inactivated poultry vaccines activate dendritic cells were explored.
Knowledge about the induction of T cell-mediated immunity by inactivated poultry vaccines is still limited. The study of Chapter 6 describes a T cell proliferation assay to evaluate cellular immunity in laying hens vaccinated with an inactivated vaccine against IBV and NDV and its potential as a quality test for this vaccine.
In Chapter 7, a review describes the difficulties that are being encountered during the transition from in vivo to in vitro batch testing of vaccines for both human and veterinary use, as well as possibilities to overcome these difficulties. Topics include the critical quality attributes of vaccines that require testing, the use of cell-based assays to mimic relevant aspects of in vivo vaccine-induced immune responses, the difficulties of adjuvanted vaccines and how to overcome these, the use of altered batches to validate in vitro test methods when non-compliant vaccine batches are limitedly available, and how cooperation between different stakeholders is key to move the transition forward. Finally, the findings of this thesis, as well as the next steps required for the transition in vitro potency testing of inactivated poultry vaccines, are discussed in Chapter 8.
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