Characterization of a hypoallergenic recombinant Bet v 1 variant as a candidate for allergen-specific immunotherapy.
BACKGROUND
Recombinant allergens and especially their hypoallergenic variants are promising candidates for a more effective and safer specific immunotherapy.


METHODS
Physicochemical and immunological characteristics of a folding variant of recombinant Bet v 1 (rBet v 1-FV) were investigated in comparison to natural Bet v 1 (nBet v 1) and the correctly folded recombinant Bet v 1 (rBet v 1-WT) by SDS-PAGE, size exclusion chromatography, multi-angle light scattering, circular dichroism, immunoblotting and enzyme allergosorbent test inhibition assay for detection of IgE reactivity and ELISA with Bet v 1-specific monoclonal antibodies. The functional IgE reactivity of the different Bet v 1 proteins was investigated using basophil activation in terms of CD203c expression and histamine release. T cell reactivity was investigated using T cell lines raised from birch pollen-allergic subjects against nBet v 1. Immunogenicity was investigated in mice.


RESULTS
Physicochemical characterization revealed purity, homogeneity and monomeric properties of rBet v 1-FV. Unlike nBet v 1 and rBet v 1-WT, rBet v 1-FV showed almost no IgE binding in immunoblots. The reduction of allergenicity was further proved by IgE-binding inhibition assays, basophil activation and histamine release. T cell reactivity was completely conserved, as demonstrated by proliferation of Bet v 1-specific T cell lines with multiple epitope specificities. rBet v 1-FV showed strong immunogenicity in mice.


CONCLUSIONS
Due to its reduced IgE reactivity and decreased capacity to activate basophils, but retained T cell reactivity and strong immunogenicity, rBet v 1-FV proved to be a very promising candidate for specific immunotherapy in birch pollen-allergic subjects.
Kahlert
H
H
Allergopharma Joachim Ganzer KG, Reinbek, Germany. helga.kahlert@allergopharma.de
Suck
R
R
Weber
B
B
Nandy
A
A
Wald
M
M
Keller
W
W
Cromwell
O
O
Fiebig
H
H
D000293:Adolescent; D000328:Adult; D000368:Aged; D000485:Allergens / Q000276:immunology* / Q000378:metabolism / Q000627:therapeutic use; D000818:Animals; D000918:Antibody Specificity; D001491:Basophils / Q000276:immunology; D029662:Betula / Q000276:immunology*; D003888:Desensitization, Immunologic / Q000379:methods*; D004353:Drug Evaluation, Preclinical; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D011058:Pollen / Q000276:immunology*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000276:immunology* / Q000378:metabolism / Q000627:therapeutic use; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology / Q000628:therapy; D013601:T-Lymphocytes / Q000276:immunology
D000485:Allergens; D000939:Epitopes; D007106:Immune Sera; D011994:Recombinant Proteins; D007073:Immunoglobulin E