title: Protection of IgE-mediated allergic sensitization by active immunization with IgE loops constrained in GFP protein scaffold.
abstract: Green fluorescent protein (GFP) exhibits a rigid central beta-barrel, formed by eleven beta-strands with floppy loops spanning between the stands. Herein, we evaluate whether the rigid beta-barrel may serve as a scaffold that can constrain the loops of a foreign protein, and thus its antigenicity. The spanning loops, site 6 of GFP, were engineered with RE cloning sites for inserting oligonucleotides corresponding to FcepsilonRI-binding sequence of human IgE. In a high-throughput format, shortened oligonucleotides encoding eight amino acid residues of the receptor-binding regions were inserted into site 6 of GFP by PCR, followed by enabling sequences for in vitro transcription and translation at the 5' end. Antigenized C2-3 linker (C2-3L) was shown by immuno-blots with polyclonal anti-IgE under native gel electrophoresis and transfer. Recombinant antigenized GFP was expressed and purified to homogeneity by metal affinity column, followed by Sephacryl S-200 high resolution gel filtration. Hyperimmune sera from mice immunized with C2-3L antigenized GFP contain anti-IgE reactive with JW8 murine/human chimeric IgE. Further, elevated serum anti-C2-3L and affinity pure antibodies effectively inhibits binding of JW8 IgE to recombinant FcepsilonRIalpha, and desensitizes JW8 to rat RBL-2H3 transfected with human FcepsilonRIalpha. This observation raised the possibility that active IgE vaccine may be employed in raising active protective anti-IgE in allergic patients as an alternative to passive immunization with MAb-E25 anti-IgE. Taken together, GFP appears suitable protein scaffold for spanning/constraining the C2-3L of human IgE as active vaccine; and this technique may be generally employed for eliciting antibodies to specific B-cell epitopes of other proteins.
authors.affiliation: Department of Allergy and Immunology, The Institute of Genetics, San Diego, CA 92121, USA. alexchen@igecure.com
authors.forename: Swey-Shen
authors.initials: SS
authors.lastname: Chen
authors.forename: Teresa
authors.initials: T
authors.lastname: Barankiewicz
authors.forename: Yong-Min
authors.initials: YM
authors.lastname: Yang
authors.forename: Maurizio
authors.initials: M
authors.lastname: Zanetti
authors.forename: Paul
authors.initials: P
authors.lastname: Hill
mesh_terms: D000818:Animals; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006967:Hypersensitivity / Q000276:immunology* / Q000517:prevention & control; D007114:Immunization / Q000379:methods*; D007073:Immunoglobulin E / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007139:Immunoglobulin epsilon-Chains / Q000276:immunology; D008407:Mast Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008968:Molecular Conformation; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D014162:Transfection
chemical_list: D018985:Epitopes, B-Lymphocyte; D007139:Immunoglobulin epsilon-Chains; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D007073:Immunoglobulin E