Protection of IgE‐mediated allergic sensitization by active immunization with antigenized IgE B‐cell epitope in GFP protein scaffold

Abstract

Green fluorescent protein (GFP) exhibits a rigid central β‐barrel, formed by eleven β‐strands with floppy loops spanning between the stands. The spanning loop, the site 6 of GFP, was engineered with RE cloning sites for inserting oligonucleotides corresponding to FcεRI‐binding sequence of human IgE. In a high throughput format, oligonucleotides encoding eight amino acids of the receptor‐binding regions were inserted in an expression cassette was assembled with 5’ in vitro transcription and translation enabling sequences. Antigenized C2‐3 linker (C2‐3L) was shown to react strongly by immunoblotting with polyclonal anti‐IgE under native gel electrophoresis and transfer. Recombinant C2‐3L antigenized GFP, expressed in recombinant vector was 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 exhibit high titers of 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 FcεRIα. and desensitizes JW8 to rat RBL‐2H3 transfected with human FcεRIα. This observation raises the distinct possibility that active IgE vaccine may indeed be employed in raising active protective anti‐IgE in allergic patients as an alternative to passive immunization with MAb‐E25 anti‐IgE. Moreover, this antigenized epitope (s) platform may be extended to analyzing specific B‐cell epitopes of other proteins of interest.