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Allergen-specific immunotherapy is the only allergen-specific and disease-modifying treatment for allergy. The construction and characterization of a vaccine for birch pollen allergy is reported. Two nonallergenic peptides, PA and PB, derived from the IgE-reactive areas of the major birch pollen allergen Bet v 1 were fused to the hepatitis B surface protein, PreS, in four recombinant fusion proteins containing different numbers and combinations of the peptides. Fusion proteins expressed in Escherichia coli and purified to homogeneity showed a lack of IgE reactivity and allergenic activity when tested with sera and basophils from patients allergic to birch pollen. Compared to Bet v 1 allergen, peptides PA and PB showed reduced T cell activation in PBMCs from allergic patients, whereas PreS fusion proteins induced less IL-5 and more IL-10 and IFN-γ. Immunization of rabbits with the fusion proteins, in particular with a PreS fusion protein 2PAPB-PreS, containing two copies of each peptide, induced high levels of IgG Abs against the major IgE-reactive site on Bet v 1 and related allergens. These IgG Abs inhibited allergic patients’ IgE binding to Bet v 1 better than did IgG induced by immunization with complete Bet v 1. Furthermore, 2PAPB-PreS–induced IgG inhibited Bet v 1–induced basophil activation in allergic patients and CD23-facilitated allergen presentation. Our study exemplifies novel beneficial features for a PreS carrier–based peptide vaccine for birch pollen, which, in addition to the established reduction in allergenic activity, include the enhanced focusing of blocking Ab responses toward IgE epitopes, immunomodulatory activity, and reduction of CD23-facilitated allergen presentation.

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients’ IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients’ IgE binding to Bet v 1 (52–75%) were obtained with mAbs specific for two peptides comprising aa 29–58 (P2) and aa 73–103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1–33, 1–57, and 31–110 of the major timothy grass pollen allergen Phl p 6 aa 1–110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical α-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the α helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31–110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31–110 inhibited patients’ IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.

House dust mites are one of the most important allergen sources. Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 are the clinically most important house dust mite (HDM) allergens. The aim of this study was to define a mix of non-allergenic T cell epitope-containing peptides of these allergens for tolerance induction. According to the amino acid sequences of these allergens, we synthesized and purified 33 overlapping peptides covering the complete sequences of Der p 1, 2, 5, 7, 21 and 23. The peptides were tested for IgE and IgG reactivity with sera from HDM allergic patients in ELISA. PBMCs from 27 HDM allergic and 10 non-HDM allergic individuals were incubated with the synthetic peptides and T cell proliferation was measured using a CFSE dilution-based assay. The peptides could be purified in large amounts. They lacked secondary structure but most of them remained soluble in physiological buffers. ELISA assays indicated that most peptides from Derp 1, 2, 5, 7, 21 and 23 lacked IgE reactivity and thus were non-allergenic. T cell proliferation assays identified 12 predominant epitopes in the Der p allergens. Our data indicates that a reasonable number of non-allergenic peptides including the sequences and thus T cell epitopes of the clinically most relevant house dust mite allergens can be defined for prevention of HDM allergy by tolerance induction.