title: Altered IgE epitope presentation: A model for hypoallergenic activity revealed for Bet v 1 trimer.
abstract: In order to reduce side effects in the course of allergen specific immunotherapy hypoallergenic allergen derivatives with reduced IgE reactivity have been made by genetic engineering. In contrast to other recombinant hypoallergenic allergen derivatives which showed reduced IgE reactivity, a recombinant trimer of the major birch pollen allergen Bet v 1 showed reduced allergenic activity despite preserved IgE reactivity. We studied rBet v 1 trimer by SDS-PAGE, mass spectrometry, circular dichroism and gel filtration. Furthermore we investigated IgE and IgG reactivity of the rBet v 1 trimer in solid and liquid phase assays and compared its allergenic activity with that of rBet v 1 wildtype using basophil activation assays. In solid phase immunoassays rBet v 1 trimer exhibited even stronger IgE reactivity than the rBet v 1 wildtype, whereas both proteins were equally well recognized by Bet v 1-specific IgG antibody probes. In fluid phase IgE experiments rBet v 1 trimer inhibited IgE reactivity to rBet v 1 wildtype but showed a more than 10-fold reduced allergenic activity compared to the rBet v 1 monomer. By analytical gel filtration it was demonstrated that, despite its monomeric appearance in SDS-PAGE the trimer occurred in fluid phase in the form of defined high molecular weight (>600 kDa) aggregates whereas rBet v 1 wildtype strictly appeared as monomeric protein. The results indicate that the hypoallergenic nature of the rBet v 1 trimer is due to formation of defined high molecular weight aggregates which may be responsible for an altered presentation of IgE epitopes in a form with reduced capacity to crosslink effector-cell bound IgE. We thus provide evidence for a novel mechanism for hypoallergenic activity.
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Vienna General Hospital (AKH), Medical University of Vienna, 1090 Vienna, Austria.
authors.forename: Raffaela
authors.initials: R
authors.lastname: Campana
authors.forename: Susanne
authors.initials: S
authors.lastname: Vrtala
authors.forename: Bernhard
authors.initials: B
authors.lastname: Maderegger
authors.forename: Yuliya
authors.initials: Y
authors.lastname: Dall'Antonia
authors.forename: Domen
authors.initials: D
authors.lastname: Zafred
authors.forename: Katharina
authors.initials: K
authors.lastname: Blatt
authors.forename: Harald
authors.initials: H
authors.lastname: Herrmann
authors.forename: Margarete
authors.initials: M
authors.lastname: Focke-Tejkl
authors.forename: Ines
authors.initials: I
authors.lastname: Swoboda
authors.forename: Sandra
authors.initials: S
authors.lastname: Scheiblhofer
authors.forename: Anna
authors.initials: A
authors.lastname: Gieras
authors.forename: Angela
authors.initials: A
authors.lastname: Neubauer
authors.forename: Walter
authors.initials: W
authors.lastname: Keller
authors.forename: Peter
authors.initials: P
authors.lastname: Valent
authors.forename: Josef
authors.initials: J
authors.lastname: Thalhamer
authors.forename: Susanne
authors.initials: S
authors.lastname: Spitzauer
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D017951:Antigen Presentation / Q000276:immunology*; D052179:Antigens, Plant / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D001491:Basophils / Q000201:enzymology / Q000378:metabolism; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005434:Flow Cytometry; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology*; D007073:Immunoglobulin E / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology; D018448:Models, Immunological; D010727:Phosphoric Diester Hydrolases / Q000276:immunology; D020836:Protein Structure, Quaternary; D011755:Pyrophosphatases / Q000276:immunology; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D012996:Solutions; D015854:Up-Regulation; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C427944:ENPP3 protein, human; D000939:Epitopes; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D012996:Solutions; D007073:Immunoglobulin E; D010727:Phosphoric Diester Hydrolases; D001619:beta-N-Acetylhexosaminidases; D011755:Pyrophosphatases