An IgE epitope of Bet v 1 and fagales PR10 proteins as defined by a human monoclonal IgE.
BACKGROUND
Analyses of the molecular basis underlying allergenicity and allergen cross-reactivity, as well as improvement of allergy diagnostics and therapeutics, are hampered by the lack of human monoclonal IgE antibodies and knowledge about their epitopes. Here, we addressed the consecutive generation and epitope delineation of a human monoclonal IgE against the prototypic allergen Bet v 1.


METHODS
Phage-display scFv hybrid libraries of allergic donor-derived VH epsilon and synthetic VL were established from 107 mononuclear cells. An obtained scFv was converted into human immunoglobulin formats including IgE. Using variants of Bet v 1, the epitope of the antibody was mapped and extrapolated to other PR10 proteins.


RESULTS
The obtained antibodies exhibited pronounced reactivity with Bet v 1, but were not reactive with the homologous PR10 protein Mal d 1. The epitope as defined by the IgE paratope and a set of chimeric Bet v 1 fusion proteins and fragments could be assigned to a C-terminal helix-structured motif comprised by amino acid residues 132-154, including the critical residue E149. Grafting this motif re-established the reactivity of the per se nonreactive Mal d 1 framework. Cross-reactivities predicted by primary structure analyses of different isoforms and PR10 proteins were verified by allergen chip-based analyses.


CONCLUSIONS
The obtained results demonstrate that hybrid IgE repertoires represent a source for human antibodies with genuine paratopes. The IgE-derived information about the IgE epitope nature of Bet v 1 and homologues allows for detailed insights into molecular aspects of allergenicity and cross-reactivity within the PR10 protein family.
Department of Chemistry, Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany.
J
J
Hecker
A
A
Diethers
D
D
Schulz
A
A
Sabri
M
M
Plum
Y
Y
Michel
M
M
Mempel
M
M
Ollert
T
T
Jakob
S
S
Blank
I
I
Braren
E
E
Spillner
D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D052179:Antigens, Plant / Q000737:chemistry / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D029964:Fagus / Q000276:immunology*; D015723:Gene Library; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment
D000485:Allergens; D052179:Antigens, Plant; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D010940:Plant Proteins; D011994:Recombinant Proteins; C061312:Bet v 1 allergen, Betula; D007073:Immunoglobulin E