Mutational analysis of the IgE epitopes in the latex allergen Hev b 5

Background: Hev b 5 is a major latex allergen and potential candidate for a n immunotherapy reagent. Objective: The purpose of this study was to produce a hypoallergenic form o f Hev b 5. Methods: We used SPOTs analysis with alanine substitution to identify amino acids (AAs) critical for IgE binding and used site-directed mutagenesis to produce recombinant proteins with altered IgE-binding activity. Results: Eleven epitopes were identified (5.1-5.11) in Hev b 5. Individual patients demonstrated variable epitope recognition, with the most intense r eactivity to epitopes 5.4 and 5.7. IgE inhibition assays with synthetic pep tides indicated that mutating a single epitope would not reduce IgE binding , but rather a combination of epitopes was required. After alanine substitu tions to identify the important AAs, site-directed mutagenesis was used to replace the crucial AAs with alanine, Twenty clones with different combinat ions of altered epitopes were evaluated by means of IgE inhibition assays. Clones with mutations in single epitopes failed to reduce IgE binding, but changes to 8 epitopes (14 AAs) resulted in a 4500-fold reduction in IgE bin ding. Epitopes 5.7 and 5.9 were found to be cross-reactive, making Hev b 5 a multivalent allergen. Conclusions: We produced a recombinant Hev b 5 protein with significantly r educed IgE-binding activity. Changing a minimum of 3 immunodominant epitope s was required to cause a 100-fold reduction in IgE binding. Changes in 8 e pitopes, particularly the cross-reactive epitopes 5.7 and 5.9, were needed to maximize the reduction in IgE binding. Mutants with reduced IgE-binding activity may prove to be valuable reagents for immunotherapy.