Anna Gieras

Mapping of Conformational IgE Epitopes with Peptide-Specific Monoclonal Antibodies Reveals Simultaneous Binding of Different IgE Antibodies to a Surface Patch on the Major Birch Pollen Allergen, Bet v 1

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.

A Nonallergenic Birch Pollen Allergy Vaccine Consisting of Hepatitis PreS–Fused Bet v 1 Peptides Focuses Blocking IgG toward IgE Epitopes and Shifts Immune Responses to a Tolerogenic and Th1 Phenotype

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.

Hypoallergenic derivatives of the major birch pollen allergen Bet v 1 obtained by rational sequence reassembly

BACKGROUND At least 100 million patients suffer from birch pollen allergy. OBJECTIVE Rational design of recombinant derivatives of the major birch pollen allergen, Bet v 1, characterized by reduced IgE reactivity, preservation of sequences relevant for the induction of allergen-specific blocking IgG, and maintenance of T-cell epitopes for immunotherapy of birch pollen allergy. METHODS Three recombinant mosaic proteins derived from Bet v 1 were generated by reassembly of codon-optimized genes coding for Bet v 1 fragments containing the elements for the induction of allergen-specific blocking IgG antibodies and the major T-cell epitopes. The proteins were expressed in Escherichia coli as recombinant mosaic molecules and compared with the Bet v 1 wild-type protein by chemical and structural methods, regarding IgE-binding and IgG-binding capacity, in basophil activation assays and tested for the in vivo induction of IgG responses. RESULTS Three recombinant Bet v 1 (rBet v 1) mosaic proteins with strongly reduced IgE reactivity and allergenic activity were expressed and purified. Immunization with the recombinant hypoallergens induced IgG antibodies that inhibited IgE reactivity of patients with allergy to Bet v 1 comparable to those induced with the rBet v 1 wild-type allergen. CONCLUSION We report the generation and preclinical characterization of 3 hypoallergenic rBet v 1 derivatives with suitable properties for immunotherapy of birch pollen allergy.

Prediction of IgE-binding epitopes by means of allergen surface comparison and correlation to cross-reactivity

BACKGROUND The experimental determination of conformational allergen epitopes recognized by IgE is a difficult task because they often involve discontinuous amino acid residues, being separated in the primary allergen sequence, and require the correct allergen fold. OBJECTIVE We sought to develop a computational tool for the localization of conformational IgE epitopes by using a structure-based comparison of allergen surfaces and IgE cross-reactivity data. METHODS Our approach involves the quantitative analysis of geometric and physicochemical surface parameters and the subsequent correlation of surface similarity scores to immunologic data. The software tool Surface comparison-based Prediction of Allergenic Discontinuous Epitopes (SPADE) is able to predict the IgE epitopes of an allergen given the availability of at least 2 structural models and IgE reactivity data. RESULTS We report on the application of our tool to 3 allergen families: the lipocalins, the group 10 pathogenesis-related proteins, and the group 2/3 grass pollen allergens. First, we succeeded in the partial relocalization of IgE epitopes of bovine β-lactoglobulin and grass pollen Phl p 2 as known from the x-ray structures of their antibody complexes. Second, we measured the relative binding of anti-Bet v 1 IgE to 10 homologous proteins and correlated these data to surface comparison results involving Bet v 1, 5 of the homologs, and 2 hypoallergenic Bet v 1 isoforms. Thereby we predicted IgE-reactive surface portions in agreement with IgE epitope-mapping studies. CONCLUSION Our approach is the first for the prediction of IgE epitopes by combining structural and IgE cross-reactivity data. It should be useful for the development of point-mutated or structurally disrupted allergen derivatives for allergen-specific immunotherapy.

Allergen cleavage by effector cell-derived proteases regulates allergic inflammation

The key event of allergic inflammation, allergen‐induced crosslinking of mast cell‐bound IgE antibodies, is accompanied by release of inflammatory mediators, cytokines, and proteases, in particular β‐tryptase. We provide evidence that protease‐mediated cleavage of allergens represents a mechanism that regulates allergen‐induced mast cell activation. When used in molar ratios as they occur in vivo, purified β ‐tryptase cleaved major grass and birch pollen allergens, resulting in defined peptide fragments as mapped by mass spectrometry. Tryptase‐cleaved allergens showed reduced IgE reactivity and allergenic activity. The biological relevance is demonstrated by the fact that lysates from activated human mast cells containing tryptase levels as they occur in vivo cleaved allergens. Additionally, protamine, an inhibitor of heparin‐dependent effector cell proteases, augmented allergen‐induced release of mediators from effector cells. Protease‐mediated allergen cleavage may represent an important mechanism for terminating allergen‐induced effector cell activation.—Rauter, I., Krauth, M.‐T., Flicker, S., Gieras, A., Westritschnig, K., Vrtala, S., Balic, N., Spitzauer, S., Huss‐Marp, J., Brockow, K., Darsow, U., Ring, J., Behrendt, H., Semper, H., Valent, P., Valenta, R. Allergen cleavage by effector cell‐derived proteases regulates allergic inflammation. FASEB J. 20, E61–E69 (2006)

Carrier‐bound nonallergenic Der p 2 peptides induce IgG antibodies blocking allergen‐induced basophil activation in allergic patients

More than 90% of house dust mite‐allergic patients are sensitized to the major Dermatophagoides pteronyssinus allergen, Der p 2. The aim of this study was to develop and characterize an allergy vaccine based on carrier‐bound Der p 2 peptides, which should allow reducing IgE‐ and T‐cell‐mediated side‐effects during specific immunotherapy (SIT).

Altered IgE epitope presentation: A model for hypoallergenic activity revealed for Bet v 1 trimer.

In order to reduce side effects in the course of allergen specific immunotherapy hypoallergenic allergen derivatives with reduced IgE reactivity have been made by genetic engineering. In contrast to other recombinant hypoallergenic allergen derivatives which showed reduced IgE reactivity, a recombinant trimer of the major birch pollen allergen Bet v 1 showed reduced allergenic activity despite preserved IgE reactivity. We studied rBet v 1 trimer by SDS-PAGE, mass spectrometry, circular dichroism and gel filtration. Furthermore we investigated IgE and IgG reactivity of the rBet v 1 trimer in solid and liquid phase assays and compared its allergenic activity with that of rBet v 1 wildtype using basophil activation assays. In solid phase immunoassays rBet v 1 trimer exhibited even stronger IgE reactivity than the rBet v 1 wildtype, whereas both proteins were equally well recognized by Bet v 1-specific IgG antibody probes. In fluid phase IgE experiments rBet v 1 trimer inhibited IgE reactivity to rBet v 1 wildtype but showed a more than 10-fold reduced allergenic activity compared to the rBet v 1 monomer. By analytical gel filtration it was demonstrated that, despite its monomeric appearance in SDS-PAGE the trimer occurred in fluid phase in the form of defined high molecular weight (>600 kDa) aggregates whereas rBet v 1 wildtype strictly appeared as monomeric protein. The results indicate that the hypoallergenic nature of the rBet v 1 trimer is due to formation of defined high molecular weight aggregates which may be responsible for an altered presentation of IgE epitopes in a form with reduced capacity to crosslink effector-cell bound IgE. We thus provide evidence for a novel mechanism for hypoallergenic activity.

IgE epitope proximity determines immune complex shape and effector cell activation capacity

Background IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells. Objective To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo. Methods We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature. Results We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation. Conclusions Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex–mediated diseases.