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.

BackgroundIgE-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.ObjectiveTo 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.MethodsWe 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.ResultsWe 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.ConclusionsOur 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.

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.

The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a ‘gatekeeper’ amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60 000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the ‘oxyanion hole’ in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE‐like enzymes. Analysis of flavin‐dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. Database Structural data are available in PDB database under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVE, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVH, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVJ, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVK, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PWB, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PWC and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PZF .