Modification of peanut allergen Ara h 3: Effects on IgE binding and T cell stimulation

Pat Rabjohn, C. Michael West, Cathie Connaughton, Hugh A. Sampson, Ricki M. Helm, A. Wesley Burks, Gary A. Bannon

Research output: Contribution to journalArticlepeer-review

81 Scopus citations

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 oligonucleo-tides 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.

Original languageEnglish
Pages (from-to)15-23
Number of pages9
JournalInternational Archives of Allergy and Immunology
Volume128
Issue number1
DOIs
StatePublished - 2002

Keywords

  • Immunotherapy
  • Modified allergen
  • Peanut allergy

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