title: Development of recombinant stable house dust mite allergen Der p 3 molecules for component-resolved diagnosis and specific immunotherapy.
abstract: BACKGROUND
  The allergen Der p 3 is underrepresented in house dust mite (HDM) extracts probably due to autolysis. Recombinant stable molecule of the allergen is thus needed to improve the diagnosis of allergy and the safety and efficacy of immunotherapy.
  
  
  OBJECTIVE
  The current study reports the immunological characterization of two recombinant molecules of the HDM allergen Der p 3 as useful tools for diagnosis and immunotherapy.
  
  
  METHODS
  Recombinant mature (rDer p 3) and immature (proDer p 3) Der p 3 and their corresponding S196A mutants were produced in Pichia pastoris and purified. The stability, IgE-binding capacity and allergenicity of the different proteins were analysed and compared with those of the major mite allergen Der p 1 used as a reference. Additionally, the immunogenicity of the different allergens was evaluated in a murine model of Der p 3 sensitization.
  
  
  RESULTS
  Compared to the IgE reactivity to recombinant and natural Der p 3 (nDer p 3), the mean IgE binding of patient's sera to rDer p 3-S196A (50%) was higher. The poorly binding to nDer p 3 or rDer p 3 was due to autolysis of the allergen. Contrary to Der p 3, proDer p 3 displayed very weak IgE reactivity, as measured by sandwich ELISA and competitive inhibition, rat basophil leukaemia degranulation and human basophil activation assays. Moreover, proDer p 3 induced a TH 1-biased immune response that prevented allergic response in mice but retained Der p 3-specific T-cell reactivity.
  
  
  CONCLUSIONS
  rDer p 3-S196A should be used for the diagnosis of HDM allergy elicited by Der p 3, and proDer p 3 may represent a hypoallergen of Der p 3.
authors.affiliation: Macromolécules Biologiques, Centre d'ingénierie des Protéines, Université de Liège, Liège, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Bouaziz
authors.forename: D
authors.initials: D
authors.lastname: Walgraffe
authors.forename: C
authors.initials: C
authors.lastname: Bouillot
authors.forename: J
authors.initials: J
authors.lastname: Herman
authors.forename: J
authors.initials: J
authors.lastname: Foguenne
authors.forename: A
authors.initials: A
authors.lastname: Gothot
authors.forename: R
authors.initials: R
authors.lastname: Louis
authors.forename: F
authors.initials: F
authors.lastname: Hentges
authors.forename: A
authors.initials: A
authors.lastname: Jacquet
authors.forename: A-C
authors.initials: AC
authors.lastname: Mailleux
authors.forename: A
authors.initials: A
authors.lastname: Chevigné
authors.forename: M
authors.initials: M
authors.lastname: Galleni
authors.forename: E
authors.initials: E
authors.lastname: Adam
authors.forename: M-E
authors.initials: ME
authors.lastname: Dumez
mesh_terms: D000485:Allergens / Q000276:immunology; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000276:immunology* / Q000378:metabolism; D060829:Arthropod Proteins / Q000276:immunology* / Q000378:metabolism; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D040002:Dermatophagoides pteronyssinus / Q000276:immunology*; D005260:Female; D006801:Humans; D006967:Hypersensitivity / Q000175:diagnosis* / Q000628:therapy*; D007114:Immunization; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D007167:Immunotherapy; D011485:Protein Binding; D059748:Proteolysis; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000276:immunology* / Q000378:metabolism
chemical_list: D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D016207:Cytokines; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D007073:Immunoglobulin E; C451165:Dermatophagoides pteronyssinus antigen p 3; D012697:Serine Endopeptidases
keywords: Der p 3; diagnosis; hypoallergen; inactive enzyme; recombinant mite allergen; specific immunotherapy