Abstract
Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70-86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Substitution / genetics
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Antimicrobial Cationic Peptides / genetics
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Antimicrobial Cationic Peptides / immunology*
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Antimicrobial Cationic Peptides / metabolism
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Basophils / immunology
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Basophils / metabolism
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Binding, Competitive / genetics
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Binding, Competitive / immunology
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Cell Division / genetics
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Cell Division / immunology
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Cell Line
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Desensitization, Immunologic* / methods
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Disulfides / chemistry
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Down-Regulation
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Epitopes, T-Lymphocyte / genetics
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Epitopes, T-Lymphocyte / immunology
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Epitopes, T-Lymphocyte / metabolism
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Humans
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Immunoglobulin E / biosynthesis
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Immunoglobulin E / metabolism
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Latex Hypersensitivity / diagnosis
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Latex Hypersensitivity / immunology*
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Lymphocyte Activation / genetics
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Lymphocyte Activation / immunology
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Peptide Fragments / genetics
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Peptide Fragments / immunology
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Peptide Fragments / metabolism
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Plant Lectins / genetics
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Plant Lectins / immunology*
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Plant Lectins / metabolism
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Protein Binding / genetics
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Protein Binding / immunology
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Protein Structure, Tertiary / genetics
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / genetics
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Recombinant Proteins / immunology
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T-Lymphocytes / cytology
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T-Lymphocytes / immunology*
Substances
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Antimicrobial Cationic Peptides
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Disulfides
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Epitopes, T-Lymphocyte
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Peptide Fragments
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Plant Lectins
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Recombinant Proteins
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hevein
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Immunoglobulin E