Conformational IgE-binding Epitopes of Major Peanut Allergen Ara h 2 Revealed with Chimeric 2S-albumins

RATIONALE: 2S-albumins are the most potent peanut allergens and specific IgE responses toward Ara h 2 and Ara h 6 are good predictors of clinical reactivity. Contrary to linear IgE-binding epitopes which have been extensively studied, data on conformational epitopes are limited. We investigated these conformational epitopes by generating chimeric 2S-albumins.

METHODS: Chimeric 2S-albumins were produced in E. coli by replacing Ara h 2 domains with the corresponding ones from Ara h 6. After purification and refolding, secondary and tertiary structures of the chimeric proteins were assessed by CD-spectroscopy and by competitive inhibition of IgE-binding to native 2S-albumins. Allergenicity of chimeras was further assessed by IgE-binding measurements with sera from 11 peanut-allergic patients and by degranulation assay of RBL-SX 38 cells.

RESULTS: All chimeric constructions exhibited a proper refolding since cross-reactive IgE antibodies to Ara h 2 and Ara h 6 recognized the chimera with similar affinity. Moreover, IgE-binding measurements performed with sera displaying no significant IgE cross-reactivity between 2S-albumins permitted to locate major conformational epitopes of Ara h 2 between residues 30 and 88. Requirement of these conformational epitopes for an efficient mast cell degranulation was confirmed with sera from patients sensitized only to Ara h 2 or with sera depleted of Ara h 6-specific IgE antibodies.

CONCLUSIONS: Conformational cross-reactive epitopes of Ara h 2 and Ara h 6 were not impacted in chimeric 2S-albumins. Although the profile of epitopes recognition remained highly patient specific, the major conformational epitopes specific to Ara h 2 were located in the central domain.