Modification of Peanut Allergen Ara h 3: Effects on IgE Binding and T Cell Stimulation

• Published in: International archives of allergy and immunology Vol. 128; no. 1; pp. 15 - 23
• Main Authors: Rabjohn, Pat, West, C. Michael, Connaughton, Cathie, Sampson, Hugh A., Helm, Ricki M., Burks, A. Wesley, Bannon, Gary A.
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland Karger 01.05.2002; S. Karger AG
• Subjects: Allergens - genetics; Allergens - immunology; Allergens - metabolism; Amino Acid Sequence; Antigens, Plant; Ara h 3 antigen; Arachis hypogaea; Biological and medical sciences; Blotting, Western; Host-tumor relations. Immunology. Biological markers; Humans; Hypersensitivity, Immediate - immunology; Hypersensitivity, Immediate - prevention & control; Immunoglobulin E - immunology; Immunoglobulin E - metabolism; Lymphocyte Activation - immunology; Medical sciences; Molecular Sequence Data; Mutagenesis, Site-Directed; Original Paper; Peanut Hypersensitivity - blood; Peanut Hypersensitivity - immunology; Peanut Hypersensitivity - prevention & control; Polymerase Chain Reaction; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; Seed Storage Proteins; T-Lymphocytes - immunology; Tumors; Modified allergen; Peanut allergy; Immunotherapy; Human; Immunopathology; Allergy; Peanut; Allergenicity; Treatment; Gene; Immunogenicity; Mutation; Recombinant protein; Food; Allergen
• ISSN: 1018-2438; 1423-0097
• DOI: 10.1159/000057999

Background: Peanut allergy is a major health concern due to the increased prevalence, potential severity, and chronicity of the reaction. The cDNA encoding a third peanut allergen, Ara h 3, has been previously cloned and characterized. Mutational analysis of the Ara h 3 IgE-binding epitopes with synthetic peptides revealed that single amino acid changes at critical residues could diminish IgE binding. Methods: Specific oligonucleotides were used in polymerase chain reactions to modify the cDNA encoding Ara h 3 at critical IgE binding sites. Four point mutations were introduced into the Ara h 3 cDNA at codons encoding critical amino acids in epitopes 1, 2, 3 and 4. Recombinant modified proteins were used in SDS-PAGE/Western IgE immunoblot, SDS-PAGE/Western IgE immunoblot inhibition and T cell proliferation assays to determine the effects of these changes on in vitro clinical indicators of peanut hypersensitivity. Results: Higher amounts of modified Ara h 3 were required to compete with the wild-type allergen for peanut-specific serum IgE. Immunoblot analysis with individual serum IgE from Ara-h-3-allergic patients showed that IgE binding to the modified protein decreased ∼35–85% in comparison to IgE binding to wild-type Ara h 3. Also, the modified Ara h 3 retained the ability to stimulate T cell activation in PBMCs donated by Ara-h-3-allergic patients. Conclusions: The engineered hypoallergenic Ara h 3 variant displays two characteristics essential for recombinant allergen immunotherapy; it has a reduced binding capacity for serum IgE from peanut-hypersensitive patients and it can stimulate T-cell proliferation and activation.