Dominant Epitopes and Allergic Cross-Reactivity: Complex Formation Between a Fab Fragment of a Monoclonal Murine IgG Antibody and the Major Allergen from Birch Pollen Bet v 1

Abstract

The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcεRI receptors on mast cell surfaces leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16, that has been solved to 2.9 Å resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients’ IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 Å2 as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order. In combination with a surprisingly high inhibitory capacity of BV16 with respect to allergic patients’ serum IgE binding to Bet v 1, these observations provide experimental support for the proposal of dominant IgE epitopes located in the conserved surface areas. This model will facilitate the development of new and safer vaccines for allergen immunotherapy in the form of mutated allergens.

Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure

“…Previously, the mAb BV16 raised against Bet v 1 was shown to compete with IgE binding to Bet v 1 [10]. Since BV16 recognizes homologous pollen allergens, but surprisingly did not cross-react with the homologous food allergens Pru av 1 and Api g 1.0101 (results not shown) in spite of the highly conserved interface [7,10], a panel of mAbs was raised against Pru av 1 and Bet v 1. The screening of their reactivity focused on binding to the P-loop region.…”

“…The P-loop is one of three highly conserved regions on the surface of the Bet v 1 molecule proposed as IgE epitopes leading to cross-reactivity with pollen allergens of other trees of the Fagales order [5]. In the crystal structure of Bet v 1 in complex with a Fab fragment of the monoclonal murine IgG1 BV16, which was raised against Bet v 1 and partially inhibits binding of human IgE, the P-loop region constitutes the contact surface, with the side chain of Glu 45 located in a positively charged binding pocket forming two hydrogen bonds with the CDR3 (complementary determining region 3) of the heavy-chain variable domain [10]. These findings suggest the P-loop region around Glu 45 as one of the IgE epitopes of Bet v 1, and the introduction of four point mutations, including a mutation from Glu 45 to Ser, indeed reduced the IgE-binding capacity of Bet v 1 [11].…”

“…This prompted us to postulate the P-loop around Glu 45 as one of at least two conformational crossreactive IgE epitopes of Pru av 1 and hence a promising candidate for site-directed mutagenesis to alter the IgE-binding properties of Pru av 1 [7]. Based on mutational studies of the weakly IgEbinding isoform Bet v 1d [15], Mal d 1 [16] from apple (Malus domestica) and Api g 1.0101 [12], a region on the adjacent first and seventh β-strands around Thr 10 and Ser 112 respectively, was also proposed as a cross-reactive IgE epitope of Pru av 1 [14].…”

Correlating IgE reactivity with three-dimensional structure

“…The only method that can determine the complete structure of a B-cell epitope is preparation of a complex of a monoclonal antibody Fab fragment with its antigen, crystallization of this complex and determination of its structure, resolving those determinants recognized by the CDRs of the antibody on the native protein. Unfortunately, the only structural information about allergen Fab crystallization complexes derives from the co-crystallization of Bet v 1, the major birch (Betula verrucosa) pollen allergen, with a mouse monoclonal IgG 1 Fab fragment in a model system [5]. However, even this model epitope covering a buried surface of 931 Å 2 must be classified as discontinuous.…”

Computational tools for the study of allergens

“…Studies of stinging insect venom allergens have shown that crossreactivity between allergens having <70% sequence identity is uncommon (13). Studies of inherent allergenicity and cross-reactivity have applications in the development of allergy immunotherapies and vaccines (14)(15)(16). Clinical and functional data for common allergens have been reported extensively and public databases contain entries of allergen sequences (17,18), structures (19), or T-cell epitopes (20,21).…”