title: Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy.
abstract: BACKGROUND
  We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy.
  
  
  METHODS
  This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation.
  
  
  RESULTS
  rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner.
  
  
  CONCLUSIONS
  On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.
authors.affiliation: Stallergènes, R&D, Antony, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Batard
authors.forename: Alain
authors.initials: A
authors.lastname: Didierlaurent
authors.forename: Henri
authors.initials: H
authors.lastname: Chabre
authors.forename: Nadine
authors.initials: N
authors.lastname: Mothes
authors.forename: Laetitia
authors.initials: L
authors.lastname: Bussières
authors.forename: Barbara
authors.initials: B
authors.lastname: Bohle
authors.forename: Marie-Noëlle
authors.initials: MN
authors.lastname: Couret
authors.forename: Tanja
authors.initials: T
authors.lastname: Ball
authors.forename: Pierrick
authors.initials: P
authors.lastname: Lemoine
authors.forename: Margarete
authors.initials: M
authors.lastname: Focks Tejkl
authors.forename: Alexandre
authors.initials: A
authors.lastname: Chenal
authors.forename: Gilles
authors.initials: G
authors.lastname: Clément
authors.forename: Francis
authors.initials: F
authors.lastname: Dupont
authors.forename: Peter
authors.initials: P
authors.lastname: Valent
authors.forename: Marie-Theres
authors.initials: MT
authors.lastname: Krauth
authors.forename: Claude
authors.initials: C
authors.lastname: André
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta
authors.forename: Philippe
authors.initials: P
authors.lastname: Moingeon
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D001491:Basophils / Q000276:immunology; D029662:Betula / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006636:Histamine Release; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D011058:Pollen / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D012130:Respiratory Hypersensitivity / Q000276:immunology* / Q000517:prevention & control*; D013601:T-Lymphocytes / Q000276:immunology; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D010940:Plant Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C061312:Bet v 1 allergen, Betula