Development of wheat allergoids for the treatment of baker's asthma

Background. Occupational exposure to inhaled proteins of wheat flour may be responsible of occupational allergies, such as baker’s asthma (BA) with a prevalence of nearly 30% among workers in industries, mills and bakeries. Up-to-date no effective specific immunotherapy (SIT) is available for the treatment of BA. The main reasons are probably the co-presence of multiple allergens in wheat and the heterogeneity of patients’ sensitization profiles. The aim of the present work is the development of wheat allergoid with reduced allergenic potency still retaining immunogenic properties to be employed in SIT for the treatment of BA.Method. The salt-soluble protein fraction was extracted by PBS from a widely utilized wheat cultivar. The flour extract (FE) was subjected to chemical modification of amino-groups with 1) potassium cyanate (K), 2) succinic anhydride (SA) and 3) acetic anhydride (AA). The three modified FE (MFEs) were assayed by immunoblotting with the sera of subjects (from the north-east part of Italy) suffering from BA (N=14) and with polyclonal antibodies specific for the major allergen α-amylase inhibitors (αAIs). K-MFE and FE were used to immunize Balb/c mice (n. 5 mice per group) with Freund’s adjuvant. Antibody titres were quantified by ELISA and the binding patterns characterized by immunoblotting. Immunodetected proteins were identified by 2D electrophoresis (2DE) and mass spectrometry (MS).Results. The immunodetection experiments confirmed an overall reduction of the IgE-binding capacity of all MFEs with very close profiles, indicating that part of the epitopes involve the aminoacid lysine, while the specific binding of the anti-αAIs antibodies was minimally affected. The immunization raised in mice different antibodies with specificity for multiple wheat proteins. The binding patterns of MFEs were almost identical and reproducible except for a 14 kDa protein recognized only by the IgG of K-MFE-injected mice. Surprisingly, the protein was detected in both FE and MFE. The separation of K-MFE and FE by 2DE showed that this protein is composed by a group of polypeptides with different isoelectric points. MS identified these proteins as αAIs.Conclusions. The raising of antibodies specific to αAIs upon administration of K-MFE might have a protective role by competing with IgE in allergen-binding. Further tests, e.g. histamine release and T cell activation, will help to evaluate the capacity of MFEs to modulate the immune response.