title: Expression of a major plant allergen as membrane-anchored and secreted protein in human cells with preserved T cell and B cell epitopes.
abstract: BACKGROUND
  Expression of allergens in human cells is a prerequisite for the development of antigen-specific cell therapy in IgE-mediated allergy. We developed a strategy how the clinically relevant major grass pollen allergen Phl p 5 can be efficiently secreted or expressed on the surface of human cells with preserved allergenic activity.
  
  
  METHODS
  The cDNA of Phl p 5 was fused to a leader peptide with or without a transmembrane domain and both constructs were ligated into a mammalian expression vector. Transfection of these plasmids into human cells resulted in a membrane-anchored or secreted version of Phl p 5, respectively, as determined by ELISA or flow cytometric analysis.
  
  
  RESULTS
  Both the secreted and membrane-anchored Phl p 5 proteins bound IgE from allergic patients in an immunoblot assay and induced specific histamine release and CD203c upregulation in basophils of grass pollen-allergic patients. Proliferation of peripheral blood mononuclear cells from Phl p 5-allergic individuals was induced upon stimulation with both variants of Phl p 5 expressed in human cells similar to recombinant Phl p 5.
  
  
  CONCLUSIONS
  Secreted and membrane-anchored Phl p 5 expressed in human cells preserved B cell as well as T cell epitopes and may be used to develop and test various cell-based strategies for allergen-specific immunomodulation and to delineate the tolerance mechanisms involved therein.
authors: Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria.
authors: Ulrike
authors: U
authors: Baranyi
authors: Martina
authors: M
authors: Gattringer
authors: Alexandra
authors: A
authors: Boehm
authors: Katharina
authors: K
authors: Marth
authors: Margarete
authors: M
authors: Focke-Tejkl
authors: Barbara
authors: B
authors: Bohle
authors: Katharina
authors: K
authors: Blatt
authors: Peter
authors: P
authors: Valent
authors: Rudolf
authors: R
authors: Valenta
authors: Thomas
authors: T
authors: Wekerle
mesh_terms: D000485:Allergens / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D052179:Antigens, Plant; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology*; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D010944:Plants / Q000276:immunology / Q000378:metabolism; D006109:Poaceae / Q000276:immunology; D011058:Pollen / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012260:Ribonucleases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D014162:Transfection
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D000954:Antigens, Surface; D018985:Epitopes, B-Lymphocyte; D018984:Epitopes, T-Lymphocyte; D008565:Membrane Proteins; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; D007073:Immunoglobulin E; C102306:Phl p Vb protein, Phleum pratense; D012260:Ribonucleases