1. Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity.
- Author
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Vrtala S, Focke M, Kopec J, Verdino P, Hartl A, Sperr WR, Fedorov AA, Ball T, Almo S, Valent P, Thalhamer J, Keller W, and Valenta R
- Subjects
- Allergens administration & dosage, Allergens immunology, Animals, Down-Regulation genetics, Female, Gene Expression Regulation immunology, Humans, Immune Sera biosynthesis, Immunoglobulin E metabolism, Immunoglobulin G biosynthesis, Immunoglobulin G metabolism, Mice, Mice, Inbred BALB C, Peptide Fragments administration & dosage, Peptide Fragments chemical synthesis, Peptide Fragments genetics, Peptide Fragments immunology, Phleum genetics, Phleum immunology, Plant Proteins administration & dosage, Plant Proteins immunology, Pollen genetics, Pollen immunology, Protein Engineering methods, Protein Folding, Protein Structure, Secondary, Rabbits, Recombinant Fusion Proteins administration & dosage, Recombinant Fusion Proteins immunology, Vaccines administration & dosage, Vaccines chemical synthesis, Vaccines immunology, Allergens biosynthesis, Allergens genetics, Down-Regulation immunology, Plant Proteins chemical synthesis, Plant Proteins genetics, Recombinant Fusion Proteins chemical synthesis, Recombinant Fusion Proteins genetics, Vaccines genetics
- Abstract
On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.
- Published
- 2007
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