From phage display to plant expression: Fulfilling prerequisites for chicken oral immunotherapy against coccidiosis

The frequency and spectrum of infections with pathogens harbouring resistance to antibiotics and other drugs has dramatically increased over the last years. One of the main causes is the extensive use of antibiotics and other drugs in human and veterinary medicine. Parasites, such as Eimeria causing coccidiosis in chicken and pathogenic bacteria like Salmonellae and Campylobacter are examples of pathogens that acquired resistance. Furthermore, continuous use of drugs in diets of animals kept for human consumption increases the risk of residues in food, that possibly affect human health. These drawbacks of antimicrobial drugs have led to a demand for alternative treatments. In this thesis an alternative approach for prevention of coccidiosis in chicken is described, based on immune intervention by passively administered, plant produced, secretory IgA.As a first step, Eimeria binding IgA fragments were selected using the phage display technique. The phage display system was adapted to be used for the display of chicken Fab fragments. A newly constructed vector, named pChick3, allows straightforward cloning of chicken variable antibody domains in frame with the constant domains of the chicken light chain and the first constant domain of the IgA heavy chain. In a following step, new plant expression vectors were designed and constructed. Ten antibodies, selected from the chicken phage antibody library were then transferred to this vector system and subsequently expressed in planta as full size IgA. Upon expression of the ten selected anti- Eimeria antibodies, differences up to 500-fold in yield were observed. Several factors on translational or protein level could cause the observed differences: e.g. processing, stability, assembly and silencing. Two were tested (silencing, chain compatibility i.e. assembly) and both have an influence on the levels of expression. An explanation may be found in the combination of several factors. These observations lead to the conclusion that an extra in planta selection step is inevitable for successful integration of phage display and plant expression systems. Finally, the structure of the chicken polymeric immunoglobulin receptor was elucidated. In a fashion similar to its mammalian counterpart, this receptor transports IgA to the gut lumen forming secretory IgA. This complex is highly stable, and IgA is protected against degradation by proteases or pH-fluctuation, which makes secretory IgA the most suitable form for passive immunization. Interestingly, the chicken SC comprises only four immunoglobulin-like domains compared to five found in mammals. Thus, an integrated system for both selection and expression of immunoglobulins was developed and with the final achievement of the production of Eimeria -specific secretory IgA in plants, the prerequisites for chicken passive immune therapy were fulfilled