title: Expression and characterization of natural-like recombinant Der p 2 for sublingual immunotherapy.
abstract: BACKGROUND
  Recombinant allergens with a native conformation represent an alternative to natural extracts for immunotherapy and diagnostic purposes.
  
  
  METHODS
  We produced the Der p 2 mite allergen in Pichia pastoris and Escherichia coli. After purification by cation exchange chromatography, recombinant molecules were compared to their natural counterpart based upon structural (disulfide bonds, secondary structure, thermal stability) and immunological properties (antibody reactivity, basophil and T cell activation, tolerance induction in a murine sublingual immunotherapy model).
  
  
  RESULTS
  The Der p 2.0101 isoform was confirmed to be prevalent in Dermatophagoides pteronyssinus extracts. It was then produced as a secreted molecule in P. pastoris or refolded from E. coli inclusion bodies. The yeast-expressed rDer p 2 molecule exhibits a natural-like disulfide bridge distribution and secondary structure, whereas the E. coli-derived rDer p 2 presents some heterogeneity in cysteine bonds and a lower stability following thermal stress. The two recombinant as well as natural Der p 2 molecules exhibit comparable IgE recognition and activate basophil and CD4+ T cells. Sublingual immunotherapy of nDer p 2- sensitized mice using either one of the rDer p 2 molecules efficiently decreases airway hyperresponsiveness as well as Th2 responses.
  
  
  CONCLUSIONS
  Natural and recombinant Der p 2 molecules produced in P. pastoris and E. coli exhibit comparable immunological properties despite distinct structural features. Natural-like cysteine pairing is a critical parameter to identify stable, well-folded and homogenous proteins appropriate for immunotherapy and diagnostic purposes.
authors.affiliation: Stallergènes SA, Antony, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Bordas-Le Floch
authors.forename: Laetitia
authors.initials: L
authors.lastname: Bussières
authors.forename: Sabi
authors.initials: S
authors.lastname: Airouche
authors.forename: Aurélie
authors.initials: A
authors.lastname: Lautrette
authors.forename: Julien
authors.initials: J
authors.lastname: Bouley
authors.forename: Nathalie
authors.initials: N
authors.lastname: Berjont
authors.forename: Stéphane
authors.initials: S
authors.lastname: Horiot
authors.forename: Axelle
authors.initials: A
authors.lastname: Huet
authors.forename: Karine
authors.initials: K
authors.lastname: Jain
authors.forename: Pierrick
authors.initials: P
authors.lastname: Lemoine
authors.forename: Henri
authors.initials: H
authors.lastname: Chabre
authors.forename: Thierry
authors.initials: T
authors.lastname: Batard
authors.forename: Laurent
authors.initials: L
authors.lastname: Mascarell
authors.forename: Véronique
authors.initials: V
authors.lastname: Baron-Bodo
authors.forename: Sophie
authors.initials: S
authors.lastname: Tourdot
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Nony
authors.forename: Philippe
authors.initials: P
authors.lastname: Moingeon
mesh_terms: D000286:Administration, Sublingual; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000008:administration & dosage / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D060829:Arthropod Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D001249:Asthma / Q000276:immunology / Q000628:therapy*; D001491:Basophils / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D003888:Desensitization, Immunologic / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D007073:Immunoglobulin E / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C451163:Dermatophagoides pteronyssinus antigen p 2; D011994:Recombinant Proteins; D007073:Immunoglobulin E