A strategic approach for immunity-based selection of cross-protective Ornithobacterium rhinotracheale Antigens

Abstract

This thesis describes the successful selection and identification of cross-protective Ornithobacterium rhinotracheale antigens with vaccine potential, using a state-of-the-art vaccinology approach. A three-way strategy was applied to i) characterize the chicken immune response during O. rhinotracheale infection, ii) identify cross-protective vaccine candidates, and iii) analyse and optimise antigen delivery. By using a complementary approach of immune depletion and reconstitution it was demonstrated that the chicken antibody-mediated immunity plays a key role in cross-protection against O. rhinotracheale infection, and that the type of immunoglobulins produced upon encountering the pathogen depends on the infection route. In order to select potential cross-protective antigens, a genomic expression library was constructed and screened with various sera from cross-protected birds. Eight different cross-reactive antigens were selected: two lipoproteins with unknown function: Or02 and Or77, three hypothetical proteins with unknown function: Or03, Or04, Or98A, and three proteins with a putative function based on sequence homologies: Or01: an enzyme involved in cell metabolism, Or11: an outer membrane protein involved in cell maintenance, and Or98B: a protein involved in DNA recombination. The genes encoding the cross-reactive antigens were cloned in an expression vector, expressed by an Escherichia coli expression strain, and recombinant proteins were purified from the growth medium. Subunit vaccination of SPF-broilers with a pool of the 8 recombinant antigens in a mineral oil adjuvant conferred (cross-) protection against a challenge with homologous and heterologous O. rhinotracheale serotypes. Of all antigens, lipoprotein Or77 demonstrated to be the antigen with highest cross-protective vaccine potential. Furthermore, Or77 subunit vaccination induced a more effective cross-protective immune response in comparison to a live vaccination or bacterin vaccination strategy. The results of a first experiment indicate that lipidation of the recombinant Or77 protein increases the immunogenicity of the vaccine antigen. Increasing the intrinsic immune-stimulatory activity of an antigen can make the addition of an adjuvant unnecessary. The study described in this thesis is an important step towards the development of a new cross-protective O. rhinotracheale vaccine and future experiments will be mainly focused on the optimisation of antigen delivery. The efficiency of the applied immuny-based approach for selection of potential vaccine candidates is proven and this strategy can also be useful for vaccine development for other pathogens.