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 an immunotherapy reagent.


OBJECTIVE
The purpose of this study was to produce a hypoallergenic form of 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 reactivity to epitopes 5.4 and 5.7. IgE inhibition assays with synthetic peptides indicated that mutating a single epitope would not reduce IgE binding, but rather a combination of epitopes was required. After alanine substitutions to identify the important AAs, site-directed mutagenesis was used to replace the crucial AAs with alanine. Twenty clones with different combinations 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 binding. 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 reduced IgE-binding activity. Changing a minimum of 3 immunodominant epitopes was required to cause a 100-fold reduction in IgE binding. Changes in 8 epitopes, 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.
Beezhold
D H
DH
Laboratory of Immunobiology, Guthrie Research Institute, 1 Guthrie Square, Sayre, PA 18840, USA.
Hickey
V L
VL
Sussman
G L
GL
D000409:Alanine / Q000235:genetics; D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D052179:Antigens, Plant; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology* / Q000378:metabolism; D020315:Latex Hypersensitivity / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000485:Allergens; D052179:Antigens, Plant; D016056:Immunodominant Epitopes; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; C101800:allergen Hev b 5; D007073:Immunoglobulin E; D000409:Alanine