Birch Pollen Allergen Bet Research Articles

Allergenicity of the major birch pollen allergen Bet v 1 might arise from molecular mimicry of homologous bacterial proteins (P6210)

Abstract Certain families of allergens contain domains that bear structural and functional similarity to homologs from pathogens enabling them to sabotage host immunity by hijacking crucial innate immune pathways. We focused on members of the Bet v 1-like superfamily that share a remarkably conserved architecture, the so called Bet v 1-fold, with the aim to identify structural mimicry as one possible cause of allergenicity. Here we show that Bet v 1, the major birch pollen (BP) allergen, has functional homology with a bacterial homolog, namely TTHA0849 from Thermus thermophilus. TTHA0849 and Bet v 1 competed for the same uptake mechanism by monocyte-derived DCs (MoDCs) of BP allergic and normal donors as visualized by fluorescence microscopy. Using a pre-designed antibacterial response PCR array, we could reveal striking similarities in gene expression patterns induced by Bet v 1 in MoDCs of both donor groups to those induced by TTHA0849. Importantly, Bet v 1 and its bacterial homolog also triggered the transcription of the Th2 signature cytokines IL-4 and IL-13, as well as EGR-3, a marker of early cell activation, in MoDCs from BP allergic but not normal donors. These findings propose that conserved structural features are present on both Bet v 1 and TTHA0849 targeting the same pathways common to allergic and normal individuals. Altered signal processing and translation in predisposed allergic individuals might be a mechanism underlying the phenomenon of allergic sensitization.

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.

Engraftment of retrovirally transduced Bet v 1-GFP expressing bone marrow cells leads to allergen-specific tolerance

Molecular chimerism is a promising strategy to induce tolerance to disease-causing antigens expressed on genetically modified haematopoietic stem cells. The approach was employed successfully in models of autoimmunity and organ transplantation. Recently, we demonstrated that molecular chimerism induces robust and lasting tolerance towards the major grass pollen allergen Phl p 5.Since allergens are a group of antigens differing widely in their function, origin and structure we further examined the effectiveness of molecular chimerism using the Phl p 5-unrelated major birch pollen allergen Bet v 1, co-expressed with the reporter GFP. Besides, inhibition of CD26 was used to promote engraftment of modified stem cells.Retrovirus VSV-Betv1-GFP was generated to transduce 5-FU-mobilized BALB/c hematopoietic cells to express membrane-bound Bet v 1 (VSV-GFP virus was used as control). Myeloablated BALB/c mice received Betv1-GFP or GFP expressing bone marrow cells, pre-treated with a CD26 inhibitor. Chimerism was followed by flow cytometry. Tolerance was assessed by measuring allergen-specific isotype levels in sera, RBL assays and T-cell proliferation assays.Mice transplanted with transduced BMC developed multi-lineage molecular chimerism which remained stable long-term (>8 months). After repeated immunizations with Bet v 1 and Phl p 5 serum levels of Bet v 1-specific antibodies (IgE, IgG1, IgG2a, IgG3 and IgA) remained undetectable in Betv1-GFP chimeras while high levels of Phl p 5-specific antibodies developed. Likewise, basophil degranulation was induced in response to Phl p 5 but not to Bet v 1 and specific non-responsiveness to Bet v 1 was observed in proliferation assays.These data demonstrate successful tolerization towards Bet v 1 by molecular chimerism. Stable long-term chimerism was achieved under inhibition of CD26. These results provide evidence for the broad applicability of molecular chimerism as tolerance strategy in allergy.

Non-Detectable IgE Binding of an Amb a 1 Derived, Contiguous Overlapping Peptide Based, SIT Product Candidate Against Ragweed Allergy

A novel approach to SIT based on Contiguous Overlapping Peptides (COPs) derived from the major birch pollen allergen Bet v 1 (AllerT™, Anergis SA, Switzerland) , has been tested successfully in a phase I/IIa clinical trial. The same technology has been applied to ragweed allergy.

Breeding of hypoallergenic strawberry fruit

BACKGROUND: Allergy to food is a hypersensitivity disorder of the immune system to normally harmless food ingredients. A promising solution for allergenic patients is the development of hypoallergenic food. OBJECTIVE: Selection and breeding of low-allergenic variety is the conventional strategy to produce hypoallergenic food. The strawberry fruit proteins Fra a 1.01E, Fra a 1.02 and Fra a 1.03 are homologous of the major birch pollen allergen Bet v1 but their individual allergenic potentials are unknown. METHOD: We produced the recombinant Fra a allergens and evaluated their cross allergenic potential in birch pollen allergic patients by a basophil activation test. Anti-Fra a 1.02 antibodies were also used to screen for allergen deficient strawberry lines. RESULTS: Although Fra a 1.01E, Fra a 1.02 and Fra a 1.03 have sequence similarities of 70, 71 and 74% with Bet v 1 Fra a 1.02 showed the highest allergenic potential. The data support the role of Fra a 1.02 as the major allergen for individuals affected by a strawberry allergy. The screening of strawberry varieties detected genotypes with significantly reduced levels of the allergen. CONCLUSION: Genotypes with reduced Fra a 1.02 proteins might serve as starting material for the breeding of hypoallergenic strawberry varieties.

Preparation and properties evaluation of the recombinant analogue of birch pollen allergen Bet v 1

Background. For wide introduction of the component allergy diagnostics in laboratory practice it is necessary to create methods of production, purification and verification of synthetic allergens. Major birch pollen protein Bet v 1, a representative of the superfamily PR-10, is interesting due to the high incidence of sensitization and the manifestation of cross-reactive properties. Methods. To receive a synthetic protein DNA cloning techniques were used, followed by expression in E. coli. Purification was made by metal-affinity chromatography. Evaluation of immunochemical properties was held in several tests including ELISA analysis and line-blot. Results. We have obtained high-yield bacterial strain E. coli, containing expression vector for protein Bet v 1 production. Physico-chemical and immunological properties were estimated in various test systems with the reference panel of serum. Conclusion. It was shown that the resulting synthetic analogue of protein Bet v 1 can be used to measure the presence of specific immunoglobulin E in serum samples. This allergenic component may be used for estimation of each component of the sensitization profile.

Passive immunization with allergen-specific IgG antibodies for treatment and prevention of allergy

IgE antibody-mediated allergies affect more than 25% of the population worldwide. To investigate therapeutic and preventive effects of passive immunization with allergen-specific IgG antibodies on allergy in mouse models we used clinically relevant pollen allergens. In a treatment model, mice were sensitized to the major birch pollen allergen Bet v 1 and to the major grass pollen allergens, Phl p 1 and Phl p 5 and then received passive immunization with rabbit IgG antibodies specific for the sensitizing or an unrelated allergen. In a prevention model, mice obtained passive immunization with allergen-specific rabbit IgG before sensitization. Kinetics of the levels of administered IgG antibodies, effects of administered allergen-specific IgG on allergen-specific IgE reactivity, the development of IgE and IgG responses and on immediate allergic reactions were studied by ELISA, rat basophil leukaemia degranulation assays and skin testing, respectively. Treated mice showed an approximately 80% reduction of allergen-specific IgE binding and basophil degranulation which was associated with the levels of administered allergen-specific IgG antibodies. Preventive administration of allergen-specific IgG antibodies suppressed the development of allergen-specific IgE and IgG1 antibody responses as well as allergen-induced basophil degranulation and skin reactivity. Our results show that passive immunization with allergen-specific IgG antibodies is effective for treatment and prevention of allergy to clinically important pollen allergens in a mouse model and thus may pave the road for the clinical application of allergen-specific antibodies in humans.

A hypoallergenic variant of the major birch pollen allergen shows distinct characteristics in antigen processing and T‐cell activation

BM4 is a novel genetically engineered variant of the major birch pollen allergen Bet v 1 that lacks the typical Bet v 1-like fold and displays negligible IgE-binding but strong T cell-activating capacity. The aim of this study was to elucidate possible differences between BM4 and Bet v 1 in internalization, antigen processing, and presentation. Proliferative responses to BM4 and Bet v 1 of peripheral blood mononuclear cells and Bet v 1-specific T-cell clones were compared. Fluorescently labeled BM4 and Bet v 1 were used to study surface binding, endocytosis, and intracellular degradation by monocyte-derived DC (mdDC). Both proteins were digested by endolysosomal extracts of mdDC. BM4- and Bet v 1-pulsed mdDC were employed to assess the kinetics of activation of Bet v 1-specific T-cell clones and the polarization of naïve T cells. BM4 displayed a significantly stronger T cell-activating capacity than Bet v 1. Furthermore, BM4 showed increased surface binding and internalization as well as faster endolysosomal degradation compared with Bet v 1. BM4-pulsed mdDC induced enhanced proliferative responses at earlier time-points in Bet v 1-specific T-cell clones and promoted less IL-5 production in T cells than Bet v 1-pulsed mdDC. The loss of the Bet v 1-fold changes the protein's interaction with the human immune system at the level of antigen-presenting cells resulting in altered T-cell responses. By combining low IgE-binding with strong and modulating T cell-activating capacity, BM4 represents a highly interesting candidate for specific immunotherapy of birch pollen allergy.

Colonoscopic allergen provocation test with rBet v 1 in patients with pollen‐associated food allergy

After consumption of fruits, nuts, and vegetables, several patients with pollen allergy experience gastrointestinal (GI) tract symptoms that are possibly caused by pollen-associated food allergy. The aim of this study was to evaluate the colonoscopic allergen provocation (COLAP) test using the recombinant birch pollen allergen Bet v 1 (rBet v 1) for in vivo diagnosis of pollen-associated food allergy manifesting in the GI tract. Thirty-four patients with a history of adverse reactions to food, GI tract symptoms, and birch pollen pollinosis and five healthy controls underwent COLAP test. Twenty minutes after endoscopic challenge of the cecal mucosa with rBet v 1, the mucosal wheal and flare reaction was registered semiquantitatively, and tissue biopsy specimens were examined for eosinophil mucosal activation. The mucosal reaction to rBet v 1 was correlated with the presence of pollinosis (P=0.004), history of adverse reaction to Bet v 1-associated food allergens (P=0.001), and tissue eosinophils' activation (P<0.001). A wheal and flare reaction in the COLAP test was observed in 13 of 16 patients (81%) with a history of GI tract symptoms associated with the ingestion of Bet v 1-related foods and in four of 18 (22%) patients with a negative history (P<0.001). The control group did not develop visible mucosal reactions to rBet v 1. Systemic anaphylactic reactions did not occur. The mucosal administration of rBet v 1 by COLAP test provides a new diagnostic tool that might support the diagnosis of Bet v 1-associated food allergy manifesting in the GI tract.

Oral exposure to Mal d 1 affects the immune response in patients with birch pollen allergy

Antibodies and T cells specific for the major birch pollen allergen Bet v 1 cross-react with structurally related food allergens, such as Mal d 1 in apple. We sought to evaluate the effects of oral uptake of Mal d 1 on the allergen-specific immune response in patients with birch pollen allergy. Patients received 50 μg of rBet v 1 sublingually on 2 consecutive days outside of the birch pollen season. One year later, equal amounts of rMal d 1 were administered. Blood samples were collected before and after oral exposure, as well as before and after the intermediate birch pollen season. Allergen-specific IgE levels were determined by using ImmunoCAP. Proliferation of allergen-stimulated PBMCs was assessed, as well as the expression of IL-5, IL-13, IL-10, IFN-γ, and forkhead box protein 3 (Foxp3) in isolated T cells (real-time PCR). Allergen-specific T-cell lines were analyzed for epitope recognition. Orally administered Bet v 1 transiently reduced Bet v 1-specific serum IgE levels, as well as Bet v 1- and Mal d 1-induced T-cell proliferation, and enhanced the expression of IL-5, IL-10, and Foxp3. Orally applied Mal d 1 significantly decreased Bet v 1- and Mal d 1-specific IgE levels and induced IL-5 and IL-10 but no Foxp3 expression. In contrast to Bet v 1, Mal d 1 triggered IFN-γ production and T cells with a different epitope repertoire. Inhalation of birch pollen significantly enhanced allergen-specific IgE levels, T-cell proliferation, and IL-5, IL-10, IL-13, and Foxp3 expression. Two sublingual administrations of 50 μg of Mal d 1 were well tolerated and induced transient immune responses seen during peripheral tolerance development. Thus recombinant Mal d 1 might be suitable and relevant for sublingual treatment of birch pollen-related apple allergy.

Crystallographically mapped ligand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1.

The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.

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

Exposure to allergens is pivotal in determining sensitization and allergic symptoms in individuals. Pollen grain counts in ambient air have traditionally been assessed to estimate airborne allergen exposure. However, the exact allergen content of ambient air is unknown. We therefore monitored atmospheric concentrations of birch pollen grains and the matched major birch pollen allergen Bet v 1 simultaneously across Europe within the EU-funded project HIALINE (Health Impacts of Airborne Allergen Information Network).Pollen count was assessed with Hirst type pollen traps at 10 l min−1 at sites in France, United Kingdom, Germany, Italy and Finland. Allergen concentrations in ambient air were sampled at 800 l min−1 with a Chemvol® high-volume cascade impactor equipped with stages PM > 10 μm, 10 μm > PM > 2.5 μm, and in Germany also 2.5 μm > PM > 0.12 μm. The major birch pollen allergen Bet v 1 was determined with an allergen specific ELISA. Bet v 1 isoform patterns were analyzed by 2D-SDS-PAGE blots and mass spectrometric identification. Basophil activation was tested in an FcɛR1-humanized rat basophil cell line passively sensitized with serum of a birch pollen symptomatic patient.Compared to 10 previous years, 2009 was a representative birch pollen season for all stations. About 90% of the allergen was found in the PM > 10 μm fraction at all stations. Bet v 1 isoforms pattern did not vary substantially neither during ripening of pollen nor between different geographical locations. The average European allergen release from birch pollen was 3.2 pg Bet v 1/pollen and did not vary much between the European countries. However, in all countries a >10-fold difference in daily allergen release per pollen was measured which could be explained by long-range transport of pollen with a deviating allergen release. Basophil activation by ambient air extracts correlated better with airborne allergen than with pollen concentration.Although Bet v 1 is a mixture of different isoforms, its fingerprint is constant across Europe. Bet v 1 was also exclusively linked to pollen. Pollen from different days varied >10-fold in allergen release. Thus exposure to allergen is inaccurately monitored by only monitoring birch pollen grains. Indeed, a humanized basophil activation test correlated much better with allergen concentrations in ambient air than with pollen count. Monitoring the allergens themselves together with pollen in ambient air might be an improvement in allergen exposure assessment.

Nitration of the Pollen Allergen Bet v 1.0101 Enhances the Presentation of Bet v 1-Derived Peptides by HLA-DR on Human Dendritic Cells

Nitration of pollen derived allergens can occur by NO2 and ozone in polluted air and it has already been shown that nitrated major birch (Betula verrucosa) pollen allergen Bet v 1.0101 (Bet v 1) exhibits an increased potency to trigger an immune response. However, the mechanisms by which nitration might contribute to the induction of allergy are still unknown. In this study, we assessed the effect of chemically induced nitration of Bet v 1 on the generation of HLA-DR associated peptides. Human dendritic cells were loaded with unmodified Bet v 1 or nitrated Bet v 1, and the naturally processed HLA-DR associated peptides were subsequently identified by liquid chromatography-mass spectrometry. Nitration of Bet v 1 resulted in enhanced presentation of allergen-derived HLA-DR-associated peptides. Both the copy number of Bet v 1 derived peptides as well as the number of nested clusters was increased. Our study shows that nitration of Bet v 1 alters antigen processing and presentation via HLA-DR, by enhancing both the quality and the quantity of the Bet v 1-specific peptide repertoire. These findings indicate that air pollution can contribute to allergic diseases and might also shed light on the analogous events concerning the nitration of self-proteins.

95 Specific Recognition of the Major Birch Pollen Allergen BET V 1 Programs Dendritic Cells to Induce Either th2 or Tolerogenic Responses

BackgroundOnly a limited number of proteins have the potential to induce Th2-polarized immune responses and specific IgE in genetically predisposed individuals. However, why the contact with an allergen results either in allergic sensitization or tolerance induction has remained unclear so far. Here, we focused on the in depth study of uptake, induction of signal transduction pathways, and gene regulation in monocyte-derived dendritic cells (MoDCs) of allergic and normal individuals in response to the major birch pollen (BP) allergen Bet v 1.0101 and its structural homolog from celery, Api g 1.0101.MethodsLive cell fluorescence microscopy was used to analyze uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs. To delineate allergen-mediated gene activation, iMoDCs were incubated with the allergens, a control stimulus, or left untreated followed by real-time PCR-based gene expression profiling. Surface-bound IgE was detected by immunofluorescence microscopy and IgE-mediated gene activation by real-time PCR.ResultsComparable kinetics of Bet v 1.0101 and Api g 1.0101 uptake were observed for both allergic and healthy donors. In competititve binding assays, however, Bet v 1.0101 outcompeted Api g 1.0101 for surface recognition in both donor groups. Pharmacological inhibition evidenced that Bet v 1.0101 internalization occurred in a receptor-mediated manner showing characteristics of lipid raft-dependent endocytosis. MoDCs of both donor groups were IgE positive and showed marked upregulation in NF-κB dependent genes after Fcε receptor activation by anti-IgE. Bet v 1.0101-stimulation, in contrast, exclusively triggered transcription of the Th2 cytokines IL-4 and IL-13 but not NF-κB related genes in MoDCs of BP allergic donors. Healthy donors were either unresponsive or showed elevated mRNA levels of the Th1-promoting chemokines CXCL10 and CXCL11.ConclusionsThis study supported by grant SFB-F1802 of the Austrian Science Fund shows for the first time that the allergen uptake is specific and similar in DCs from allergic and healthy individuals. More important, the ensuing signal cascade that is triggered by the allergen differs between DCs of the 2 donor groups and results in a Th2-polarized immune response in allergic individuals as compared to ignorance/tolerance in normal donor cells.

23 Grafting of BET V 1 Epitopes onto its Homologue API G 1 Reveals Patient-Specific IgE Recognition Profiles

BackgroundUp to 70% of birch pollen-allergic individuals show adverse reactions to certain plant foods. This cross-reactivity is caused by sensitisation to the major birch pollen allergen Bet v 1 and binding of Bet v 1-specific IgE antibodies to homologous plant food allergens. We aimed to assess the importance of selected conformational epitopes for IgE binding to Bet v 1.MethodsChimeras of Bet v 1.0101 and its homologue Api g 1.0101 were constructed. In each of the 4 chimeras, roughly one fourth of the surface residues of Api g 1.0101 were replaced by corresponding residues of Bet v 1.0101. The proteins were expressed in Escherichia coli and purified by chromatographic methods. Secondary structures were checked by CD-spectroscopy. IgE ELISA with Bet v 1.0101, Api g 1.0101 and the chimeras were performed with sera of 63 Bet v 1-sensitized birch pollen allergic patients. For inhibition ELISAs, chimeras were coated and inhibition was performed with the chimeras or Api g 1.0101.ResultsIgE binding to Api g 1.0101, Api-Bet-1, -2, -3 and -4 was observed for 22, 81, 79, 70 and 38% of the sera, respectively. To assess the relevance of the grafted regions for IgE binding to Bet v 1, the amounts of IgE binding to the chimeras were compared with those to Api g 1.0101. Most of the sera recognised either 3 chimeras (39%) or all 4 chimeras (21%) better than Api g 1.0101. Only a minority of the sera showed increased binding to a single chimera. Inhibition ELISAs confirmed the presence of IgE specific for the grafted regions.ConclusionsOur study indicates that the epitope recognition profile of Bet v 1-specific IgE is highly patient specific. Due to the different IgE binding patterns to Bet v 1, determined by binding of IgE to different chimeras, the existence of a single major IgE epitope on Bet v 1 can be excluded. Moreover, the Bet v 1-specific IgE repertoire is polyclonal and the IgE epitopes are distributed over the whole surface of Bet v 1.

Human blood basophils do not act as antigen‐presenting cells for the major birch pollen allergen Bet v 1

Several studies in mice have recently shown that basophils can act as antigen-presenting cells (APC) inducing Th2-mediated immune responses against parasites or protease allergens. The aim of this study was to investigate whether human basophils function as APC for the major birch pollen allergen Bet v 1. Fluorescently labeled Bet v 1 was used to assess surface binding and internalization of allergen by basophils and different types of APC from birch pollen-allergic and nonallergic individuals. Sorted basophils were analyzed in terms of up-regulation of MHC class II and co-stimulatory molecules in the absence and presence of IL-3 and IFN-γ by flow cytometry. Expression of proteins crucial for antigen presentation, namely cathepsin S and invariant chain, was determined. Basophils were used as APC in co-culture experiments with Bet v 1-specific T-cell clones (TCCs). Basophils from birch pollen-allergic donors very efficiently bound Bet v 1 through IgE/FcεRI complexes on their surface. In contrast to professional APC, basophils did not internalize allergen and expressed marginal levels of cathepsin S and invariant chain. HLA-DP, HLA-DQ, CD80/CD86, and CD40 were absent from purified basophils even when stimulated with IL-3 plus IFN-γ. IL-3/IFN-γ marginally up-regulated HLA-DR. Bet v 1-pulsed basophils failed to induce proliferative and cytokine responses in Bet v 1-specific, HLA-DR-restricted TCCs. Human basophils neither internalize, process nor present Bet v 1. Because Bet v 1 is a highly relevant allergen, we conclude that basophils play no role as APC in IgE-mediated allergy in humans.

Human TCR Transgenic Bet v 1-Specific Th1 Cells Suppress the Effector Function of Bet v 1-Specific Th2 Cells

Pollinosis to birch pollen is a common type I allergy in the Northern Hemisphere. Moreover, birch pollen-allergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological cross-reactivity. The major T cell epitope Bet v 1(142-153) plays an important role in cross-reactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this study, we cloned and functionally analyzed a human αβ TCR specific for the immunodominant epitope Bet v 1(142-153). cDNAs encoding TCR α- and β-chains were amplified from a Bet v 1(142-153)-specific T cell clone, introduced into Jurkat T cells and peripheral blood T lymphocytes of allergic and nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCRtg) T cells responded in an allergen-specific and costimulation-dependent manner to APCs either pulsed with Bet v 1(142-153) peptide or coexpressing invariant chain::Bet v 1(142-153) fusion proteins. TCRtg T cells responded to Bet v 1-related food and tree pollen allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1(142-153)-presenting but not Bet v 1(4-15)-presenting artificial APCs coexpressing membrane-bound IL-12 polarized allergen-specific TCRtg T cells toward a Th1 phenotype, producing high levels of IFN-γ. Coculture of such Th1-polarized T cells with allergen-specific Th2-differentiated T cells significantly suppressed Th2 effector cytokine production. These data suggest that human allergen-specific TCR can transfer the fine specificity of the original T cell clone to heterologous T cells, which in turn can be instructed to modulate the effector function of the disease initiating/perpetuating allergen-specific Th2-differentiated T cells.

Anaphylaktische Reaktion nach Sojagenuss bei einem Patienten mit Birkenpollenallergie

Patients with birch pollen allergy may suffer from severe anaphylactic reactions after ingestion of foodstuffs containing soya. The reason for this is similarities in protein structure between a major birch pollen allergen (bet v 1) and Gly m 4, a pollen-related protein in soya. A 65-year-old patient allergic to birch pollen developed an adverse systemic reaction after consumption of a soya-containing drink. The diagnosis could be confirmed by in vitro and skin testing methods. Patients who suffer from birch pollen allergy should strictly avoid the intake of soya-containing foodstuffs.

Extraction and mass spectrometry identification of a major peach allergen Pru p 1

Peach allergy can be caused by the allergen Pru p 1. This occurs by cross-reactivity with the homologous birch pollen allergen Bet v 1. However, the direct identification of Pru p 1 as an immunoglobulin E (IgE)-binding protein extracted from peach fruit has never been reported. Phosphate-buffered saline (PBS) and phenol extractions were applied to solubilise the proteins from peach peel and pulp, and IgE immunoblotting with sera of individual peach-allergic patients was used to detect the potential allergens. Most of the patients showed binding to an 18 kDa band in IgE immunoblotting performed with the phenolic extracts of peach peel and pulp, but not when the PBS extracts were used. Mass spectrometry of the 18 kDa spot excised from a two-dimensional electrophoretic gel showed this protein to correspond to the peach allergen Pru p 1. Phenol extraction was necessary to detect by IgE immunoblotting a major peach allergen, which showed very low extractability with PBS, indicating the appropriateness of adopting different extraction procedures to identify plant allergens. The 18 kDa peach protein was definitively identified as the Bet v 1-homologous peach allergen Pru p 1.

Introductory lecture: Pollen food allergy syndrome

The term pollen-food allergy syndrome (PFAS) defines a series of clinical symptoms appearing shortly after the ingestion of plant-derived foods in subjects with pollen allergy. The patients with PFAS are primarily allergic to pollen and subsequently react to food allergens as a consequence of the homology between pollen and plant-food proteins. The two highly conserved proteins responsible for the large majority of cases of pollen-food allergy syndrome are the pathogenesis-related proteins group 10 (PR-10), including the major birch pollen allergen Bet v 1 and homologous proteins in different fruits and vegetables, and profilin, a plant pan-allergen present in cell structure of all the vegetable kingdom. Although it has been generally thought that the clinical expression of the pollen food-allergy syndrome is uniquely the so-called “oral allergy syndrome”, recent reports suggest that the ingestion of particular foods may be associated with systemic symptoms as well. Recombinant PR-10 proteins and recombinant profilins from different sources are presently available for diagnostic purposes. The presentation will review the available data about the clinical expression, diagnosis, and therapy of the pollen-food allergy syndrome.