title: Modification of peanut allergen Ara h 3: effects on IgE binding and T cell stimulation.
abstract: 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 approximately 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Arkansas Children's Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72202, USA.
authors.forename: Pat
authors.initials: P
authors.lastname: Rabjohn
authors.forename: C Michael
authors.initials: CM
authors.lastname: West
authors.forename: Cathie
authors.initials: C
authors.lastname: Connaughton
authors.forename: Hugh A
authors.initials: HA
authors.lastname: Sampson
authors.forename: Ricki M
authors.initials: RM
authors.lastname: Helm
authors.forename: A Wesley
authors.initials: AW
authors.lastname: Burks
authors.forename: Gary A
authors.initials: GA
authors.lastname: Bannon
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D015153:Blotting, Western; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology / Q000517:prevention & control; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021183:Peanut Hypersensitivity / Q000097:blood / Q000276:immunology / Q000517:prevention & control; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D055314:Seed Storage Proteins; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; C118207:allergen Ara h3; D007073:Immunoglobulin E