Modification of peanut allergen Ara h 3: effects on IgE binding and T cell stimulation.
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.
Rabjohn
Pat
P
Department of Biochemistry and Molecular Biology, Arkansas Children's Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72202, USA.
West
C Michael
CM
Connaughton
Cathie
C
Sampson
Hugh A
HA
Helm
Ricki M
RM
Burks
A Wesley
AW
Bannon
Gary A
GA
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
D000485:Allergens; D052179:Antigens, Plant; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; C118207:allergen Ara h3; D007073:Immunoglobulin E