PMU-Autor/inn/en
Hartl Arnulf JosefAbstract
The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phi p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost-their structural fold. Allergic patientsxxx IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patientsxxx IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergenxxxs three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.
Useful keywords (using NLM MeSH Indexing)
Allergens/chemistry
Allergens/genetics*
Allergens/immunology
Allergens/metabolism
Animals
Anti-Allergic Agents/administration*
dosage
Anti-Allergic Agents/chemical synthesis
Anti-Allergic Agents/immunology
Antigens, Plant
Basophils/immunology
Basophils/secretion
Binding Sites, Antibody/genetics
Binding, Competitive/immunology
Calcium-Binding Proteins/chemistry
Calcium-Binding Proteins/genetics*
Calcium-Binding Proteins/immunology
Calcium-Binding Proteins/metabolism
Cell Degranulation
Cell Line, Tumor
Cross Reactions
Desensitization, Immunologic/methods*
Dose-Response Relationship, Immunologic
Histamine Release/immunology
Humans
Immunoglobulin E/metabolism
Immunoglobulin G/biosynthesis
Immunoglobulin G/metabolism
Mice
Mutagenesis, Site-Directed
Peptide Fragments/administration*
dosage
Peptide Fragments/genetics
Peptide Fragments/immunology
Peptide Fragments/metabolism
Phleum/chemistry
Phleum/genetics
Phleum/immunology*
Plant Proteins/chemistry
Plant Proteins/genetics*
Plant Proteins/immunology
Plant Proteins/metabolism
Pollen/genetics
Pollen/immunology
Rabbits
Rats
Skin Tests
Structure-Activity Relationship
Vaccines/administration*
dosage
Vaccines/chemical synthesis*
Vaccines/genetics*
Vaccines/immunology