Recombinant egg allergens as specific immunotherapeutic agents of egg allergy

This thesis is an investigation of developing non-mutated glycosylated recombinant ovalbumin, and designing and developing recombinant ovalbumin mutants; testing their protective efficiency against anaphylaxis in a mouse model; investigating their mechanisms of action against anaphylaxis in this model; and investigating the mechanism of action of recombinant ovomucoid third domain in oral specific immunotherapy. Recombinant technology and bioinformatics offer powerful tools to create molecules effective against complex diseases, such as allergies, as well as for further studies, including the study of the mechanisms of action of molecules. Non-mutated glycosylated recombinant ovalbumin was developed in Pichia pastoris, and eight site-directed recombinant ovalbumin mutants were designed and developed in P. pastoris / Escherichia coli. Among the recombinant molecules tested in mice through oral specific immunotherapy, a double mutant V58D / K280E Pp and a triple mutant R59G / K280E / F3075 Ec - H were effective in completely preventing anaphylaxis. In these mice there was an increase in expression of cell surface markers CD4, CD25 and TCR-gammadelta on lymphocytes from intestinal Peyer's patch, and on intraepithelial and lamina propria lymphocytes, together with an increase in production of TGF-beta. This increased the secretion of IgA and induced the Treg cell cytoplasmic marker / transcription factor FoxP3 expression which along with increased IL-10, as well as changes in the levels and expression of other cytokines and transcription fators, skewed the Th1 / Th2 balance towards the Th1 pathway. Taken together, V58D / K280E Pp and R59G / K280E / F307S Ec - H play a key role 1) in skewing the Teff/ Treg balance towards the Treg pathway, and 2) in skewing the Th1 / Th2 balance towards the Th1 pathway. These mutants may serve as potential therapeutic agents against ovalbumin/egg allergy and as safe potential substitutes for ovalbumin in food products.;Recombinant ovomucoid third domain tested through oral specific immunotherapy in mice resulted in lower levels of immunoglobulins, as well as lower expression of cytokines and transcription factors than V58D / K280E Pp and R59G / K280E / F307S Ec - H, suggesting a similar mechanism of action but with weaker attributes.