Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01.

The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1. IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant). Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404. It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen. Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.