Four Recombinant Isoforms of Cor a 1, the Major Allergen of Hazel Pollen, Show Different Reactivities with Allergen‐specific T‐lymphocyte Clones

Abstract

Purified preparations of allergenic proteins from plants, and in particular from pollens, consist of multiple closely related isoforms. These isoforms are highly similar in their amino acid sequences, yet they display different properties with respect to antibody binding. In this study we report of differential potencies of cross-reacting tree pollen allergens and cloned isoforms of these allergens to activate allergen-specific T-lymphocyte clones (T-cell clones ; TCC). Six TCC with specifity for Bet v 1, a representative tree pollen major allergen, were established from peripheral blood of five birch-pollen-allergic donors. All TCC displayed the helper-cell phenotype. Five TCC reacted with distinct epitopes present on natural (n) and on recombinant (r) Bet v 1. One TCC could not be stimulated with r Bet v 1, in spite of strong reactivity with purified natural Bet v 1. The TCC were tested in proliferation assays using purified n Bet v 1, n Cor a 1 (the homologous major allergen of hazel pollen), r Bet v 1, four recombinant isoforms of Cor a 1 and peptides representing corresponding T-cell stimulating regions (isoepitopes) on these proteins. The clones showed different patterns of reactivity in response to stimulation with the five recombinant molecules and the corresponding peptides. Certain exchanges of amino acids within stimulating peptides correlated with a lack of proliferation of the TCC tested. These findings are important with respect to the use of broadly cross-reactive recombinant allergens or allergen-derived peptides for immunotherapy of type I allergy.Type I allergy to tree pollens is very common in the northern hemisphere. In this respect, pollens from trees of the order Fagales, in particular birch, hazel and alder, represent the most important sources of allergens [l, 21. The major allergens of these pollens (Bet v 1, Cor a 1 and A h g 1) show a high degree of similiarity at the nucleic acid 131 and at the amino acid levels [4], and cross-reactivity was described for IgE antibodies [5] and for T-cell clones (TCC) [6]. This fits well with the observation that tree pollen-allergic patients develop symptoms when exposed to pollens from all these tree species.Natural allergens consist of multiple isoallergens [7 -91. Recently, four isoforms of Cor a 1, the major allergen of hazel pollen, were cloned, sequenced and expressed as recombinant non-fusion proteins [8]. Comparison of the amino acid sequences of these Cor a 1 isoforms revealed identities Correspondence to

Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy.

“…Birch pollen allergen, Bet v 1 (9,12), was chosen as a model because it represents the major allergen for Ͼ 95% of tree pollen and plant food allergic patients and Ͼ 60% of these patients are sensitized exclusively to Bet v 1 (12,13). In addition, Bet v 1 binds a high percentage of specific serum IgE antibodies and shares B cell as well as T cell epitopes with homologous allergens present in tree pollen and plant-derived food (15,16,(31)(32)(33)(34).…”

“…The cDNA coding for Bet v 1 has been isolated recently (9) and recombinant Bet v 1 was expressed in Escherichia coli (13,14). Recombinant Bet v 1 has been shown to possess an IgE-binding capacity similar to that of natural Bet v 1 and shares IgE as well as T cell epitopes with Bet v 1 homologous proteins present in pollen from various trees and in plantderived food (6,15,16). The biological activity of recombinant Bet v 1 has been demonstrated by histamine release experiments and by skin prick testing of allergic patients (17)(18)(19).…”

The Importance of Recombinant Allergens for Diagnosis and Therapy of IgE–Mediated Allergies

“…Our findings showing that Bet v 1 and other closely related tree pollen allergens consist of a mixture of closely related isoforms displaying striking differences in their ability to bind IgE and activate allergen–specific T cell clones [25, 26, 27]led us to speculate that these differences resulted from only a few amino acid sequence differences. To analyze the patterns of amino acid exchanges in all tree pollen isoallergens and their IgE–binding activities, we used an algorithm recently developed by Casari et al [28]to predict functional residues in proteins.…”

Genetically Engineered Plant Allergens with Reduced Anaphylactic Activity

“…The allergenic integrity of recombinant Bet v 1 (rBet v 1) was shown in in vitro and in vivo experiments. Not only does rBet v 1 possess an IgE–binding capacity comparable to the native protein, but it also retains the IgE epitopes that the native protein shares with other allergens [23, 58, 59]. rBet v 1 has also been shown to stimulate Bet v 1–specific T–cell clones [23], induce the release of histamine from basophils [60]and produce an immediate–type wheal reaction in skin prick tests [61, 62].…”