title: Immune responses after immunization with plasmid DNA encoding Bet v 1, the major allergen of birch pollen.
abstract: BACKGROUND
  Immunization with plasmid DNA encoding various antigens is a promising method in vaccine research. Recent studies also indicate that DNA-based immunization might represent a potential approach in allergen-specific immunotherapy.
  
  
  OBJECTIVE
  In this study we have characterized the immune responses induced by recombinant Bet v 1a and plasmid DNA encoding for Bet v 1a, the major allergen of birch pollen in a mouse system.
  
  
  METHODS
  Balb/c mice were injected intraperitoneally with recombinant Bet v 1a and intradermally with plasmid DNA encoding for the gene of Bet v 1a (pCMV-Bet). In addition, the effect of immunostimulatory DNA sequences was investigated by appending CpG motifs to the gene of Bet v 1a, coinjecting CpG-oligodeoxynucleotides together with the pCMV-Bet construct, or both. IgE and IgG antibody responses, as well as IgG subclasses, were measured by ELISA in sera after each immunization. IFN-gamma and IL-4 levels were also measured by ELISA in sera and supernatants of allergen-stimulated spleen cells.
  
  
  RESULTS
  The primary humoral response to a single treatment with pCMV-Bet was very weak, but the reaction could be boosted to higher levels by 2 additional injections. On the other hand, proliferation assays of spleen cells and measurements of cytokine levels already indicated a cellular response after the first injection of plasmid DNA. After 2 immunizations with pCMV-Bet, the ratio of IgG1 to IgG2a pointed to a TH1 subclass profile. IgE was not detectable in any group at any time during the immune reaction. Accordingly, IL-4 levels were markedly reduced in the serum, as well as in the supernatants, of stimulated spleen cells. Animals immunized with pCMV-Bet containing appended CpG motifs at the 3' end of the Bet v 1a gene and/or with the CpG-ODN GCTAGACGTTAGCGT plus pCMV-Bet displayed reduced humoral responses against Bet v 1a when compared with animals injected with pCMV-Bet alone. The levels of IFN-gamma measured after allergen stimulation of isolated spleen cells were significantly higher in animals immunized with pCMV-Bet plus CpG motifs than with pCMV-Bet alone. Immunization with recombinant Bet v 1a protein elicited a strong TH2 -type response, including IgE production, a high titer of IgG1, and IL-4 production in both serum and supernatants of proliferation cultures.
  
  
  CONCLUSIONS
  In contrast to immunization with protein, DNA immunization induces a strong TH1 -type response against a relevant inhalant allergen. Our data support the concept of developing a novel type of allergen immunotherapy based on plasmid DNA immunization.
authors.affiliation: Institute of Chemistry and Biochemistry, University of Salzburg, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Hartl
authors.forename: J
authors.initials: J
authors.lastname: Kiesslich
authors.forename: R
authors.initials: R
authors.lastname: Weiss
authors.forename: A
authors.initials: A
authors.lastname: Bernhaupt
authors.forename: S
authors.initials: S
authors.lastname: Mostböck
authors.forename: S
authors.initials: S
authors.lastname: Scheiblhofer
authors.forename: C
authors.initials: C
authors.lastname: Ebner
authors.forename: F
authors.initials: F
authors.lastname: Ferreira
authors.forename: J
authors.initials: J
authors.lastname: Thalhamer
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology; D000818:Animals; D000917:Antibody Formation / Q000187:drug effects; D052179:Antigens, Plant; D002455:Cell Division; D018899:CpG Islands / Q000502:physiology; D016207:Cytokines / Q000097:blood; D004247:DNA / Q000276:immunology*; D005260:Female; D007114:Immunization; D007167:Immunotherapy; D051379:Mice; D008807:Mice, Inbred BALB C; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology*; D010957:Plasmids / Q000235:genetics*; D013154:Spleen / Q000166:cytology; D018417:Th1 Cells / Q000502:physiology
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D016207:Cytokines; D010940:Plant Proteins; C061312:Bet v 1 allergen, Betula; D004247:DNA