Dulashi Withanage Dona

Hypersensitivity or an allergy to chicken egg proteins is a predominant symptomatic condition affecting 1 in 20 children in Australia; however, an effective form of therapy has not yet been found. This occurs as the immune system of the allergic individual overreacts when in contact with egg allergens (egg proteins), triggering a complex immune response. The subsequent instantaneous inflammatory immune response is characterized by the excessive production of immunoglobulin E (IgE) antibody against the allergen, T-cell mediators and inflammation. Current allergen-specific approaches to egg allergy diagnosis and treatment lack consistency and therefore pose safety concerns among anaphylactic patients. Immunotherapy has thus far been found to be the most efficient way to treat and relieve symptoms, this includes oral immunotherapy (OIT) and sublingual immunotherapy (SLIT). A major limitation in immunotherapy, however, is the difficulty in preparing effective and safe extracts from natural allergen sources. Advances in molecular techniques allow for the production of safe and standardized recombinant and hypoallergenic egg variants by targeting the IgE-binding epitopes responsible for clinical allergic symptoms. Site-directed mutagenesis can be performed to create such safe hypoallergens for their potential use in future methods of immunotherapy, providing a feasible standardized therapeutic approach to target egg allergies safely.

Background: Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges. The mutated and non-mutated proteins were expressed in Escherichia coli (E. coli) by induction with isopropyl β-d-1-thiogalactopyranoside (IPTG). The expressed proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to confirm expression. Immunoglobulin E (IgE) reactivity of the two proteins was analysed, by immunoblotting, against a pool of egg-allergic patients’ sera. A pool of non-allergic patients’ sera was also used in a separate blot as a negative control; Results: Mutant Gal d 1 showed diminished IgE reactivity in the immunoblot by showing lighter bands when compared to the non-mutated version, although there was more of the mutant protein immobilised on the membrane when compared to the wild-type protein. The non-allergic negative control showed no bands, indicating an absence of non-specific binding of secondary antibody to the proteins; Conclusion: Disruption of two cysteine bridges in domain III of Gal d 1 reduces IgE reactivity. Following downstream laboratory and clinical testing, this mutant protein can be used in immunotherapy to induce tolerance to Gal d 1 and in egg allergy diagnosis.