Characterization of a Hypoallergenic Recombinant Bet v 1 Variant as a Candidate for Allergen-Specific Immunotherapy

Abstract

Background: Recombinant allergens and especially their hypoallergenic variants are promising candidates for a more effective and safer specific immunotherapy. Methods: Physicochemical and immunological characteristics of a folding variant of recombinant Bet v 1 (rBet v 1-FV) were investigated in comparison to natural Bet v 1 (nBet v 1) and the correctly folded recombinant Bet v 1 (rBet v 1-WT) by SDS-PAGE, size exclusion chromatography, multi-angle light scattering, circular dichroism, immunoblotting and enzyme allergosorbent test inhibition assay for detection of IgE reactivity and ELISA with Bet v 1-specific monoclonal antibodies. The functional IgE reactivity of the different Bet v 1 proteins was investigated using basophil activation in terms of CD203c expression and histamine release. T cell reactivity was investigated using T cell lines raised from birch pollen-allergic subjects against nBet v 1. Immunogenicity was investigated in mice. Results: Physicochemical characterization revealed purity, homogeneity and monomeric properties of rBet v 1-FV. Unlike nBet v 1 and rBet v 1-WT, rBet v 1-FV showed almost no IgE binding in immunoblots. The reduction of allergenicity was further proved by IgE-binding inhibition assays, basophil activation and histamine release. T cell reactivity was completely conserved, as demonstrated by proliferation of Bet v 1-specific T cell lines with multiple epitope specificities. rBet v 1-FV showed strong immunogenicity in mice. Conclusions: Due to its reduced IgE reactivity and decreased capacity to activate basophils, but retained T cell reactivity and strong immunogenicity, rBet v 1-FV proved to be a very promising candidate for specific immunotherapy in birch pollen-allergic subjects.

Validation of Basophil CD164 Upregulation for Pollen Allergy Diagnosis

“…Control stimulations with anti-IgE antibody and fMLP gave satisfactory results, as observed in other similar studies (Ebo et al 2005;Ebo et al 2006;Kahlert et al 2008;Monneret et al 2002). All patients positively responded with anti-IgE stimulation; in contrast 20% of controls were negative.…”

Release of Bet v 1 from birch pollen from 5 European countries. Results from the HIALINE study

“…Extracts were aliquoted, frozen, lyophilized and stored at À20 C. Aliquots were reconstituted in 0.1 M phosphate buffered saline pH7.4, serial diluted and allergen was determined by using a 2-site binding assay based on monoclonal antibodies in an ELISA format. Monoclonal antibodies 4B10 and 2E10 specific for Bet v 1, natural purified Bet v 1 standards and controls were provided by our partner Allergopharma KG, Reinbek, Germany Kahlert et al, 2008). With each ELISA two control samples of different concentration were analyzed.…”

Detection of Bet v1, Bet v2 and Bet v4 Specific IgE Antibodies in the Sera of Children and Adult Patients Allergic to Birch Pollen: Evaluation of Different IgE Reactivity Profiles Depending on Age and Local Sensitization

“…Simultaneously, it is possible to monitor the efficacy of applied immunotherapy by means of the level of specific IgE, IgG, and IgG subclasses against recombinant allergens [7]. In the future, it might be possible to use the precisely defined recombinant allergens also for individually tailored hyposensibilization [7,8,9,10]. …”