title: A mutant of the major apple allergen, Mal d 1, demonstrating hypo-allergenicity in the target organ by double-blind placebo-controlled food challenge.
abstract: BACKGROUND
  Allergen-specific immunotherapy for food allergy has been hindered by severe side-effects in the past. Well-characterized hypo-allergenic recombinant food allergens potentially offer a safe solution.
  
  
  OBJECTIVE
  To demonstrate hypo-allergenicity of a mutated major food allergen from apple, Mal d 1, in vitro and in vivo.
  
  
  METHODS
  A mutant of the major apple allergen, Mal d 1, was obtained by site-directed mutagenesis exchanging five amino acid residues. Fourteen patients with combined birch pollen-related apple allergy were included in the study. Hypo-allergenicity of the mutant rMal d 1 (rMal d 1mut) compared with rMal d 1 was assessed by in vitro methods, i.e. RAST (inhibition), immunoblotting and basophil histamine release (BHR) and in vivo by skin prick test and double-blind placebo-controlled food challenge (DBPCFC).
  
  
  RESULTS
  RAST analysis (n = 14) revealed that IgE reactivity to rMal d 1mut was twofold lower than that of the wild-type molecule (95% confidence interval (CI): 1.7-2.4). RAST inhibition (n = 6) showed a 7.8-fold decrease in IgE-binding potency (95% CI: 3.0-12.6). In contrast to this moderate decrease in IgE-binding potency, the biological activity of rMal d 1mut assessed by SPT and BHR decreased 10-200-fold. Hypo-allergenicity was confirmed by DBPCFC (n = 2) with both recombinant molecules.
  
  
  CONCLUSIONS
  A moderate decrease in IgE-binding potency translates into a potent inhibition of biological activity. This is the first study that confirms by DBPCFC that a mutated recombinant major food allergen is clinically hypo-allergenic. This paves the way towards safer immunotherapy for the treatment of food-allergic patients.
authors.affiliation: Department of Dermatology/Allergology, University Medical Center Utrecht, Utrecht, The Netherlands. s.bolhaar@azu.nl
authors.forename: S T H P
authors.initials: ST
authors.lastname: Bolhaar
authors.forename: L
authors.initials: L
authors.lastname: Zuidmeer
authors.forename: Y
authors.initials: Y
authors.lastname: Ma
authors.forename: F
authors.initials: F
authors.lastname: Ferreira
authors.forename: C A F M
authors.initials: CA
authors.lastname: Bruijnzeel-Koomen
authors.forename: K
authors.initials: K
authors.lastname: Hoffmann-Sommergruber
authors.forename: R
authors.initials: R
authors.lastname: van Ree
authors.forename: A C
authors.initials: AC
authors.lastname: Knulst
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000235:genetics* / Q000276:immunology*; D052179:Antigens, Plant; D001491:Basophils / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005512:Food Hypersensitivity / Q000276:immunology*; D030841:Food, Genetically Modified; D006636:Histamine Release; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology; D008297:Male; D027845:Malus; D008875:Middle Aged; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology*; D011864:Radioimmunosorbent Test; D012882:Skin Tests
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C095551:MALD1 protein, Malus domestica; D010940:Plant Proteins; D007073:Immunoglobulin E