title: Recombinant fusion proteins assembling Der p 1 and Der p 2 allergens from Dermatophagoides pteronyssinus.
abstract: BACKGROUND
  Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy.
  
  
  METHODS
  Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays.
  
  
  RESULTS
  Four fusion proteins were expressed in E. coli as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens.
  
  
  CONCLUSIONS
  Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.
authors.affiliation: Research and Development, Stallergènes, Antony, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Bussières
authors.forename: Véronique
authors.initials: V
authors.lastname: Bordas-Le Floch
authors.forename: Ingrid
authors.initials: I
authors.lastname: Bulder
authors.forename: Henri
authors.initials: H
authors.lastname: Chabre
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Nony
authors.forename: Aurélie
authors.initials: A
authors.lastname: Lautrette
authors.forename: Christelle
authors.initials: C
authors.lastname: Berrouet
authors.forename: Yvette
authors.initials: Y
authors.lastname: Nguefeu
authors.forename: Stéphane
authors.initials: S
authors.lastname: Horiot
authors.forename: Véronique
authors.initials: V
authors.lastname: Baron-Bodo
authors.forename: Laurence
authors.initials: L
authors.lastname: Van Overtvelt
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: De Conti
authors.forename: Anne
authors.initials: A
authors.lastname: Schlegel
authors.forename: Nicolas
authors.initials: N
authors.lastname: Maguet
authors.forename: Nicolas
authors.initials: N
authors.lastname: Mouz
authors.forename: Pierrick
authors.initials: P
authors.lastname: Lemoine
authors.forename: Thierry
authors.initials: T
authors.lastname: Batard
authors.forename: Philippe
authors.initials: P
authors.lastname: Moingeon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry / Q000276:immunology*; D060829:Arthropod Proteins; D001491:Basophils / Q000502:physiology; D003546:Cysteine Endopeptidases; D040002:Dermatophagoides pteronyssinus / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006967:Hypersensitivity / Q000628:therapy; D008213:Lymphocyte Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology; D014612:Vaccines / Q000276:immunology
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C451163:Dermatophagoides pteronyssinus antigen p 2; D011993:Recombinant Fusion Proteins; D014612:Vaccines; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1