title: Epitope specificity determines cross-protection of a SIT-induced IgG4 antibody.
abstract: BACKGROUND
  The calcium-binding 2EF-hand protein Phl p 7 from timothy grass pollen is a highly cross-reactive pollen pan-allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7-specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen-specific immunotherapy (SIT).
  
  
  METHODS
  We studied epitope specificity, cross-reactivity, affinity and cross-protection of mAb102.1F10 towards homologous calcium-binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were made with purified recombinant allergens. Binding and cross-reactivity of patients' IgE and mAb102.1F10 to calcium-binding allergens and peptides thereof were studied with quantitative RAST-based methods, in ELISA, basophil activation and IgE-facilitated allergen presentation experiments.
  
  
  RESULTS
  Allergens from timothy grass (Phl p 7), alder (Aln g 4), birch (Bet v 4), turnip rape (Bra r 1), lamb's quarter (Che a 3) and olive (Ole e 3, Ole e 8) showed high sequence similarity and cross-reacted with allergic patients' IgE. mAb102.1F10 bound the C-terminal portion of Phl p 7 in a calcium-dependent manner. It cross-reacted with high affinity with Ole e 3, whereas binding and affinity to the other allergens were low. mAb102.1F10 showed limited cross-inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross-reactivity.
  
  
  CONCLUSIONS
  Our results demonstrate that a small number of amino acid differences among cross-reactive allergens can reduce the affinity of binding by a SIT-induced IgG and thus limit cross-protection.
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Centre for Pathophysiology, Infectiology and Immunology, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.
authors.forename: E
authors.initials: E
authors.lastname: Gadermaier
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, UK.
authors.forename: L K
authors.identifier: http://orcid.org/0000-0002-2252-4636
authors.initials: LK
authors.lastname: James
authors.affiliation: Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College London, London, UK.
authors.forename: M H
authors.initials: MH
authors.lastname: Shamji
authors.affiliation: Division of Hematology and Hemostaseology, Department of Internal Medicine I, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.
authors.forename: K
authors.initials: K
authors.lastname: Blatt
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria.
authors.forename: K
authors.initials: K
authors.lastname: Fauland
authors.affiliation: Vienna Challenge Chamber, Allergy Centre Vienna West, Vienna, Austria.
authors.forename: P
authors.initials: P
authors.lastname: Zieglmayer
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Centre for Pathophysiology, Infectiology and Immunology, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.
authors.forename: T
authors.initials: T
authors.lastname: Garmatiuk
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Centre for Pathophysiology, Infectiology and Immunology, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Focke-Tejkl
authors.affiliation: Departamento de Bioquımica y Biologıa Molecular I, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Villalba
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, UK.
authors.forename: R
authors.initials: R
authors.lastname: Beavil
authors.affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria.
authors.forename: W
authors.identifier: http://orcid.org/0000-0002-2261-958X
authors.initials: W
authors.lastname: Keller
authors.affiliation: Division of Hematology and Hemostaseology, Department of Internal Medicine I, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.
authors.forename: P
authors.initials: P
authors.lastname: Valent
authors.affiliation: Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College London, London, UK.
authors.forename: S R
authors.initials: SR
authors.lastname: Durham
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, UK.
authors.forename: H J
authors.initials: HJ
authors.lastname: Gould
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Centre for Pathophysiology, Infectiology and Immunology, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Flicker
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Centre for Pathophysiology, Infectiology and Immunology, Vienna General Hospital, Medical University of Vienna, Vienna, Austria.
authors.forename: R
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D052179:Antigens, Plant; D002118:Calcium / Q000378:metabolism; D003429:Cross Reactions / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology*; D007167:Immunotherapy; D008958:Models, Molecular; D010455:Peptides / Q000276:immunology; D011058:Pollen / Q000276:immunology*; D011485:Protein Binding / Q000276:immunology; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D000939:Epitopes; D007074:Immunoglobulin G; D010455:Peptides; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D002118:Calcium
keywords: SIT-induced IgG antibody; calcium-binding protein; cross-reactivity; pollen allergen; recombinant allergen