Birch Pollen Allergic Patients Research Articles

Human Leukocyte Antigen-G and Regulatory T Cells during Specific Immunotherapy for Pollen Allergy

Background: T_H2-biased immune responses are important in allergy pathogenesis. Mechanisms of allergen-specific immunotherapy (SIT) might include the induction of regulatory T cells (Tregs) and immunoglobulin (Ig) G₄ blocking antibodies, a reduction in the number of effector cells, and skewing of the cytokine profile towards a T_H1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG₄). Methods: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG₄ were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated with pollen extract in vitro and immune factors were evaluated. Results: During SIT, the main changes in the peripheral blood were an increase in CXCR3⁺CD4⁺CD25⁺CD127^low/- Tregs and a decrease in CCR4⁺CD4⁺CD25⁺CD127^low/- Tregs, an increase in allergen-specific IgG₄, and a decrease in sHLA-G during the first half of the treatment period. In the PBMC in vitro experiments, the following changes were observed upon allergen-stimulation: an increase in CD4⁺CD25⁺CD127^low/- Tregs and ILT2⁺CD4⁺CD25⁺CD127^low/- Tregs, an increase in IL-10 and IL-2 levels, and an increase in sHLA-G that was most pronounced at the start of SIT. Conclusions: The changes in CXCR3⁺CD4⁺CD25⁺CD127^low/- Treg, IgG₄, and sHLA-G levels in the peripheral blood and in ILT2⁺ Treg, IL-10, IL-2, and sHLA-G levels upon in vitro allergen stimulation suggest an upregulation in immunomodulatory factors and, to some degree, a shift towards T_H1 during SIT.

Vig r 6, the cytokinin‐specific binding protein from mung bean (Vigna radiata) sprouts, cross‐reacts with Bet v 1‐related allergens and binds IgE from birch pollen allergic patients’ sera

ScopeBirch pollen associated allergy to mung bean sprouts is caused by cross-reactivity between the birch pollen allergen Bet v 1 and the mung bean allergen Vig r 1. We aimed to determine the allergenicity of the cytokinin-specific binding protein from mung bean (Vig r 6), another allergen related to Bet v 1 with only 31% sequence identity.Methods and resultsBet v 1, Gly m 4, Vig r 1, and Vig r 6 were produced in Escherichia coli. In an ELISA, 73 and 32% of Bet v 1-sensitized birch-allergic patients’ sera (n = 60) showed IgE binding to Vig r 1 and Vig r 6, respectively. Of 19 patients who reported allergic reactions or had positive prick-to-prick tests to mung bean sprouts, 79% showed IgE binding to Vig r 1 and 63% showed IgE binding to Vig r 6. Bet v 1 completely inhibited IgE binding to both mung bean allergens. Vig r 6 showed partial cross-reactivity with Vig r 1 and activated basophils sensitized with mung bean allergic patients’ sera.ConclusionWe demonstrated IgE cross-reactivity despite low sequence identity between Vig r 6 and other Bet v 1-related allergens. Thus, IgE binding to Vig r 6 may contribute to birch pollinosis-associated mung bean sprout allergy.

Polysensitisation to pollen due to profilin and calcium-binding protein: distribution of IgE antibodies to marker allergens in grass and birch pollen allergic rhinitis patients in southern Germany

Allergen-specific immunotherapy for grass pollen allergy has been reported to be effective in up to 85% of patients. Sensitisation to profilin and calcium-binding protein (CBP) can possibly influence treatment results and may thus be a reason for treatment failures. During a study period of 3 years, the distribution patterns of antibodies to marker allergens were continuously investigated in all blood serum samples with a level of immunoglobulin E antibodies to timothy and birch pollen higher than 0.7 kUA/l (n = 556). Sensitisation to timothy grass pollen alone was found in 33% of the cases, to birch pollen alone in 19%, and to both in 48%. The group of polysensitised patients showed an inhomogenous distribution of antibodies to marker allergens. IgE against minor allergens was detected in 40%. Sensitisation to major allergens, especially to the major birch allergen, was not present in 13% of the polysensitised patients. Of the patients who were sensitised to minor allergens, 82% were sensitised to profilin, 11% to CBP, and 8% to both profilin and CBP. Profilin and CBP frequently cause polysensitisations to pollen. The data obtained justify the measurement of serum levels of antibodies to marker allergens in patients who are sensitised to more than one group of allergens.

Generation of a protein scaffold for the analysis of functional immunoglobulin epitopes of Bet v 1-like allergens

Background Millions of patients with allergy to tree pollen are sensitized to the major allergen of birch (Betula verrucosa) pollen, Bet v 1. Bet v 1-specific IgE cross-reacts with Bet v 1-homologous proteins from plant foods. Only little information on functional IgE epitopes of Bet v 1 and Bet v 1-like allergens in foods is available. We sought to generate a synthetic protein tool to identify and analyze functional immunoglobulin binding sites on Bet v 1 and Bet v 1-like allergens. Methods A synthetic protein (synNCS) was engineered based on the primary structures of both native Norcoclaurine synthase, a non-allergenic protein of the Bet v 1 superfamily, and a natural hypoallergenic Bet v 1 variant, respectively. Recombinant synNCS was purified from Escherichia coli. Site-directed mutagenesis was applied to generate synNCS variants carrying potential IgE epitopes of Bet v 1. synNCS and variants thereof were physicochemically characterized using circular dichroism spectroscopy and dynamic light scattering. To evaluate IgE interaction with synNCS, sera of birch pollen allergic patients were tested for IgE binding to purified synNCS in Western blot, ELISA, and cellular mediator release assays. Results Recombinant synNCS variants were expressed in E. coli. The theoretical secondary structure topology is similar to Bet v 1. Very low IgE binding to synNCS could be detected in Western blot analyses. However functional in vitro IgE interaction with synNCS variants was dependent of the number of potential Bet v 1 IgE epitope residues inserted into the model protein. The synNCS variants induced IgE-mediated mediator release in humanized rat basophil leukemia cells sensitized with sera of birch pollen allergic subjects. Conclusion

Double‐blind, placebo‐controlled, dose‐ranging study of new recombinant hypoallergenic Bet v 1 in an environmental exposure chamber

Recombinant allergens offer a tool for improving specific immunotherapy (SIT). To find the optimal dose of a new hypoallergenic folding variant of recombinant Bet v 1 (rBet v 1-FV) as SIT for patients with birch pollen allergy. Before SIT, thirty-seven adult patients were exposed for eight hours in an environmental exposure chamber (EEC) to birch pollen at an average concentration of 3500 ± 500 grains/m(3) , then randomized to four maintenance dose groups of rBet v 1-FV and one placebo group: 20 μg (n = 7), 80 μg (n = 8), 160 μg (n = 7), 320 μg (n = 8), and placebo (n = 7). Patients were treated for 10 weeks with weekly injections and then re-exposed in the EEC. The optimal dose for SIT was assessed using efficacy results from the EEC, IgG responses, and tolerability. Thirty-six patients were evaluable for efficacy assessment. The total symptom score significantly decreased in all active groups compared with placebo (-18.8% for placebo patients; -71.9%, P = 0.0022 for 20 μg; -75.6%, P = 0.0007 for 80 μg; -81.8%, P = 0.0009 for 160 μg; -78.3%, P = 0.0003 for 320 μg). IgG1 increased significantly in all active groups compared to placebo. All four active doses were well tolerated, no serious adverse event occurred; two Grade II reactions, according to EAACI classification, were observed, one in each of the 160- and 320-μg groups. Considering efficacy, immunological response, and tolerability, a maintenance dose of 80 μg of rBet v 1-FV appears to be the ideal dose for allergen immunotherapy in birch pollen allergic patients.

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.

Hypoallergenic profilin – a new way to identify allergenic determinants

Plant profilins are important panallergens, contributing to numerous food and pollen allergies. They fulfil essential functions in all eukaryotic cells, including yeast and mammals, making breeding of profilin-deficient crop plants an impossible task. To obtain hypoallergenic profilin-variants, a novel yeast based screening system to select for fully active but hypoallergenic profilins was developed. Despite similar biological functions, plant profilins share only limited amino acid sequence identity to nonplant profilins. Thus, it should be possible to select for hypoallergenic profilin variants that have kept their biological function. These variants were subsequently tested for IgE-binding and their 3D folding was analyzed. As prerequisites, we developed a conditional profilin-deficient yeast mutant strain and made use of a newly-discovered profilin variant from tomato that enabled pre-selection for efficient mutagenesis. After random mutagenesis and transformation of tomato profilin (Lyc e 1) into the designed yeast strain, we were able to screen for functional tomato profilin variants. Testing these variants for IgE binding revealed that two variants showed a strong reduction in IgE binding of four Lyc e 1 sensitized patients and one birch pollen allergic patient. This result, for the first time, provides strong evidence that selection of hypoallergenic Lyc e 1 variants is possible. Furthermore, this knowledge provides the basis for the molecular breeding of hypoallergenic profilin alleles in tomato.

Prevention of Birch Pollen-Related Food Allergy by Mucosal Treatment with Multi-Allergen-Chimers in Mice

BackgroundAmong birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery) or Dau c 1 (carrot), termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy.MethodologyBALB/c mice were poly-sensitized with a mixture of Bet v 1, Api g 1 and Dau c 1 followed by a sublingual challenge with carrot, celery and birch pollen extracts. For prevention of allergy sensitization an allergen chimer composed of immunodominant T cell epitopes of Api g 1 and Dau c 1 linked to the whole Bet v 1 allergen, was intranasally applied prior to sensitization.ResultsIntranasal pretreatment with the allergen chimer led to significantly decreased antigen-specific IgE-dependent β-hexosaminidase release, but enhanced allergen-specific IgG2a and IgA antibodies. Accordingly, IL-4 levels in spleen cell cultures and IL-5 levels in restimulated spleen and cervical lymph node cell cultures were markedly reduced, while IFN-γ levels were increased. Immunomodulation was associated with increased IL-10, TGF-β and Foxp3 mRNA levels in NALT and Foxp3 in oral mucosal tissues. Treatment with anti-TGF-β, anti-IL10R or anti-CD25 antibodies abrogated the suppression of allergic responses induced by the chimer.ConclusionOur results indicate that mucosal application of the allergen chimer led to decreased Th2 immune responses against Bet v 1 and its homologue food allergens Api g 1 and Dau c 1 by regulatory and Th1-biased immune responses. These data suggest that mucosal treatment with a multi-allergen vaccine could be a promising treatment strategy to prevent birch pollen-related food allergy.

Birch pollen associated soy allergy – possibilities in diagnostic and clinical relevance1)

Abstract Background: Birch pollen allergic individuals frequently suffer from food allergies in the form of an oral allergy syndrome after eating pome and stone fruits. These complaints are based on an immunological cross-reaction between pollen and food allergens. In the past, it has been shown that many birch pollen allergic patients are additionally not able to tolerate high protein soy products. Some severe immediate type reactions to soy have been observed. The cause for these immediate type reactions to soy is a Bet v 1 cross-reactive soy allergen called Gly m 4. Methods: Using a collective of 73 birch pollen allergic patients with associated food allergy in Leipzig as an example, the results of a standardized questioning, prick-to-prick test with a soy drink, determination of specific IgE against rGly m 4, and basophil activation test with Gly m 4 are presented. Results and conclusions: We showed that commercially available prick test extracts and determination of specific IgE against soy bean mix/f14 are not appropriate to diagnose birch pollen associated soy allergy. Generally, soy sensitization could be proven when a prick-to-prick-test with a soy drink and determination of specific IgE against rGly m 4 were done. A positive prick-to-prick test with a soy drink was found in 79% (55/70) of the birch pollen allergic patients with 89% (65/73) showing specific IgE for rGly m 4 (CAP>1). Although not every sensitization was clinically relevant, every third patient with a proven soy sensitization was diagnosed with a clinically relevant allergy to soy.

471 IgE from Birch Pollen Allergic Patients Cross-reacts with Two Distinct Bet V 1 Related Proteins in Mung Beans

BackgroundMung beans (Vigna radiata) contain 2 Bet v 1 related proteins: Vig r 1, a member of the PR-10 subfamily, and cytokinin-specific binding protein (CSBP), a protein with low sequence identity (31%) to Bet v 1. We aimed to compare Vig r 1 and CSBP to Bet v 1 regarding biochemical and immunological properties.MethodsPercent surface identity between Bet v 1, CSBP and Vig r 1 was calculated based on structural alignments using an algorithm considering backbone conformations and identities of aligned residues. The allergens were expressed in Escherichia coli and purified by metal chelate affinity and ion exchange chromatography. Secondary structures were compared using circular dichroism (CD) spectroscopy. Binding and cross-reactivity of IgE from Bet v 1-sensitized patients' sera to rCSBP, rVig r 1.0101 and rBet v 1.0101 were examined by ELISA and ELISA inhibition.ResultsStructural comparison of the 3 proteins revealed that 29% of the solvent-accessible surface area of CSBP was identical to Bet v 1, while Vig r 1 and Bet v 1 shared 50% surface area. In addition, 2 surface patches, conserved between Bet v 1 and CSBP, were identified as potential cross-reactive epitopes. 30% and 79% of Bet v 1-sensitized birch pollen allergic patients' sera (n = 33) showed IgE binding to CSBP and Vig r 1, respectively. Of 12 Bet v 1-sensitized patients, who reported reactions or had positive prick-to-prick tests to mung bean sprouts, 10 showed IgE binding to Vig r 1 and 7 to CSBP. Bet v 1 completely inhibited IgE binding to CSBP and Vig r 1. Furthermore, CSBP showed inhibitory activity on IgE binding to Vig r 1 and vice versa.ConclusionsThis study demonstrates IgE cross-reactivity between Bet v 1 and CSBP, despite their low sequence identity. In addition to Vig r 1, a PR-10 subfamily member, IgE binding to CSBP might contribute to allergic reactions in mung bean sprouts.

Reaction To Soymilk But Not Other Soy Products In Gly m 4 Sensitized Birch Pollen-Allergic Patients

Reaction To Soymilk But Not Other Soy Products In Gly m 4 Sensitized Birch Pollen-Allergic Patients

Birch pollen associated soy allergy – possibilities in diagnostic and clinical relevance1)

AbstractBirch pollen allergic individuals frequently suffer from food allergies in the form of an oral allergy syndrome after eating pome and stone fruits. These complaints are based on an immunological cross-reaction between pollen and food allergens. In the past, it has been shown that many birch pollen allergic patients are additionally not able to tolerate high protein soy products. Some severe immediate type reactions to soy have been observed. The cause for these immediate type reactions to soy is a Bet v 1 cross-reactive soy allergen called Gly m 4.Using a collective of 73 birch pollen allergic patients with associated food allergy in Leipzig as an example, the results of a standardized questioning, prick-to-prick test with a soy drink, determination of specific IgE against rGly m 4, and basophil activation test with Gly m 4 are presented.We showed that commercially available prick test extracts and determination of specific IgE against soy bean mix/f14 are not appropriate to diagnose birch pollen associated soy allergy. Generally, soy sensitization could be proven when a prick-to-prick-test with a soy drink and determination of specific IgE against rGly m 4 were done. A positive prick-to-prick test with a soy drink was found in 79% (55/70) of the birch pollen allergic patients with 89% (65/73) showing specific IgE for rGly m 4 (CAP>1). Although not every sensitization was clinically relevant, every third patient with a proven soy sensitization was diagnosed with a clinically relevant allergy to soy.

STAT5 in human basophils: IL‐3 is required for its FcεRI‐mediated phosphorylation

Basophils are key effector cells in allergic inflammatory reactions. However, the mechanisms of FcεRI-induced degranulation are complex and remain to be disentangled. Signal transducer and activator of transcription (STAT) molecules modulate various cell functions. STAT5 appears to be essential for IgE-mediated mast cell function, but its role in human basophils after cross-linking FcεRI is unknown. In this study, STAT5 phosphorylation was investigated by flow cytometry, and combined with analyses of the degranulation marker CD63 at single cell level. Kinetics of STAT5 phosphorylation were studied in basophils of birch pollen allergic patients and showed a fast phosphorylation induced by interleukin (IL)-3, but not with antigen alone. Stimulating basophils with a mixture of allergen and IL-3 resulted in a two to three fold higher phosphorylation of STAT5 than induced by IL-3 alone. In the presence of IL-3, antigen elicited a dose-dependent STAT5 response. In conclusion, this study demonstrates that STAT5 in human basophils is activated through both the IL-3 and the FcεRI signaling pathway.

Ongoing seasonal intestinal inflammation in birch pollen allergic patients without gastrointestinal symptoms

Background A previous study by our group (JACI 2003;112:45-50) showed a significant allergic gastrointestinal inflammation in birch pollen allergic patients with gastrointestinal symptoms during the birch pollen season. The pathophysiological mechanisms of this type of allergic reaction are still poorly studied. However it is not yet explored if all birch pollen patients have signs of gastrointestinal inflammation during the pollen season.

Impact OF Allergen Specific Immunotherapy on Allergen Threshold for Basophil Activation and Inhibitory IgG Antibodies in Birch Pollen Allergic Patients

Impact OF Allergen Specific Immunotherapy on Allergen Threshold for Basophil Activation and Inhibitory IgG Antibodies in Birch Pollen Allergic Patients

Patients with allergic rhinitis and allergic asthma share the same pattern of eosinophil and neutrophil degranulation after allergen challenge

BackgroundPatients with allergic rhinitis and allergic asthma demonstrate comparable local and systemic eosinophil inflammation, and yet they present with different clinical pictures. Less is even known about the contribution of neutrophil inflammation in allergic diseases. The aim of the study was to examine the propensity and selectivity of granule release from primed systemic eosinophils and neutrophils in allergic rhinitis and allergic asthma after seasonal and experimental allergen exposure. We hypothesize that the dissimilar clinical manifestations are due to diverse eosinophil and neutrophil degranulation.MethodsNine birch pollen allergic patients with rhinitis, eight with asthma and four controls were studied during pollen season and after nasal and bronchial allergen challenge. Eosinophils and neutrophils were incubated in vitro with assay buffer and opsonized Sephadex particles for spontaneous and C3b-induced granule protein release. The released amount of eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) was measured by specific radioimmunoassay.ResultsC3b-induced degranulation resulted in increased release of ECP and MPO from primed blood eosinophils and neutrophils in both allergic rhinitis and allergic asthma during pollen season and after both nasal and bronchial challenge (p-values 0.008 to 0.043). After bronchial challenge, the ECP release was significantly higher in the rhinitic group compared to the asthmatic group [19.8 vs. 13.2%, (p = 0.010)]. The propensity for EPO release was weak in all challenge models but followed the same pattern in both allergic groups.ConclusionsSystemically activated eosinophils and neutrophils have similar patterns of degranulation after allergen exposure in allergic rhinitis and allergic asthma. The released amount of ECP, EPO and MPO was similar in all allergen challenge models in both allergic groups. Our results indicate that other mechanisms than the magnitude of eosinophil and neutrophil inflammation or the degranulation pattern of the inflammatory cells determines whether or not an allergic patient develops asthma.

Genetic engineering of trimers of hypoallergenic fragments of the major birch pollen allergen, Bet v 1, for allergy vaccination

An immunotherapy trial performed in allergic patients with hypoallergenic recombinant fragments, comprising aa 1–74 and 75–160 of the major birch pollen allergen, Bet v 1, has indicated that the induction of allergen-specific IgG responses may be an important mechanism of this treatment. To investigate whether the immunogenicity of the rBet v 1 fragments can be increased, recombinant trimers of the fragments were produced. For this purpose, DNA trimers of rBet v 1 aa 1–74 as well as of rBet v 1 aa 75–160 were subcloned into expression plasmid pET 17b, expressed in Escherichia coli and purified. The fragments as well as the fragment trimers showed a reduced IgE-binding capacity and allergenic activity compared to rBet v 1 wildtype when tested in allergic patients. Both rBet v 1 aa 75–160 monomer and trimer induced high titers of allergen-specific IgG1 Abs in mice. Interestingly, rBet v 1 aa 1–74 trimer induced a much higher IgG1 response to rBet v 1 than rBet v 1 aa 1–74 monomer. Consequently, IgG Abs induced with the rBet v 1 aa 1–74 trimer inhibited birch pollen allergic patients’ IgE-binding 10-fold more efficiently than IgG Abs induced with the monomer. Our data show that the immunogenicity of allergy vaccines can be increased by oligomerization.

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

Background: Birch pollen belongs to the major allergen triggers in the spring season in Europe. Our rapidly expanding knowledge of the allergenic molecules enables us to better recognize the individual differences between the reactivity of specific IgE antibodies of individual patients and allergic populations living in various regions of the world. Method: In a group of birch pollen-allergic patients living in the Czech Republic (107 children, 71 adults) we detected the presence of Bet v1, Bet v2 and Bet v4 specific IgE antibodies. Results: Bet v1 specific IgE antibodies were identified in most patients without any significant differences between children and adults. Bet v2 positivity was found more frequently in the group of children than in adults (p = 0.02). In most adult patients Bet v1 monospecificity was more expressed as compared to the pediatric group. More allergic subjects reacted against minor birch allergens in the pediatric group (p = 0.02). Specific IgE antibodies against Bet v1 were not detected in 10% of the tested patients. In this group, 5% of birch pollen-allergic patients were found to not have specific IgE antibodies against any of the tested recombinant allergens. Conclusion: The investigation of specific IgE antibodies against Bet v1, Bet v2 and Bet v4 demonstrated that the specificity of allergen-induced IgE antibodies in birch pollen-allergic individuals is dependent not only on the region in which a patient lives but also on age. Especially in children, there is an increase in the number of allergic subjects who do not react exclusively against the major allergen. The question is whether some allergen-specific IgE antibodies will disappear depending on age or on the contrary whether their synthesis will be increased.

Inhibition of CD23‐dependent facilitated allergen binding to B cells following vaccination with genetically modified hypoallergenic Bet v 1 molecules

Vaccination with hypoallergenic recombinant Bet v 1 derivatives (Bet v 1 fragments and Bet v 1 trimer) is associated with the induction of IgG antibodies specific to natural Bet v 1. To investigate whether IgG antibodies induced following vaccination with genetically modified hypoallergenic Bet v 1 derivatives are able to inhibit IgE-facilitated binding of allergen-IgE complexes to B cells. Sera from 46 patients obtained before and after subcutaneous vaccination with Bet v 1 trimer (n=14), Bet v 1 fragments (n=11) or placebo (n=21) were incubated with recombinant (r) Bet v 1 and an indicator serum (IS) from a birch pollen-allergic patient with high CD23 binding capacity. Bet v 1 immune complexes were added to a CD23-expressing B cell line and co-operative binding of Bet v1-IgE complexes to CD23 was measured with a polyclonal anti-IgE FITC antibody using a bio-functional cellular flow cytometric assay. When sera from patients vaccinated with rBet v 1 derivatives were incubated with Bet v 1 and the IS, a reduction of IgE binding to CD23 was observed. This effect was not seen when sera from placebo-treated patients were used. The decrease in CD23/IgE binding was statistically significant in the trimer group [pre- vs. post-specific immunotherapy (SIT): P=0.02; trimer vs. placebo: P<0.04] but not in the Bet v 1 fragments-treated group. Trimer-treated patients had higher levels of Bet v 1-specific IgG than fragment-treated patients. The degree of inhibitory activity of IgE-facilitated allergen binding correlated with Bet v 1-specific IgG levels following SIT (R=0.492; P=0.012). Vaccination with both recombinant Bet v 1 derivatives induces Bet v 1-specific IgG antibodies, which are able to inhibit the co-operative binding of allergen-IgE complexes to CD23, and may thereby reduce allergen-specific T cell responses.

Identification of Bet v 1‐related allergens in fig and other Moraceae fruits

Allergy to fig fruit (Ficus carica) has been described in patients allergic to Ficus benjamina or rubber latex but may occur also in pollen-allergic patients. To study the potential cross-reactivity between fig and taxonomically related fruits with the major birch pollen allergen Bet v 1. One hundred and eighty-eight patients with or without birch pollen allergy were prick-to-prick tested with fig (F. carica), mulberry (Morus alba), jackfruit (Artocarpus heterophyllus; all family Moraceae) and other pollen-associated foods. Moraceae fruit extracts were separated by SDS-PAGE and tested with patient sera and polyclonal antisera against Mal d 1. Western blot inhibition was performed with Moraceae fruit extracts, birch pollen and recombinant Bet v 1. Putative Bet v 1 homologs in Moraceae fruits were analysed by liquid chromatography-ion trap mass spectrometry. Among 85 patients with isolated birch pollen allergy, 78% had a positive skin test to fresh fig, 10% to dried fig, 91% to mulberry, 91% to jackfruit, 77% to Rosaceae fruits and 83% to hazelnut. Sixty-six per cent of birch pollen-allergic patients positive for fig, reported symptoms after consumption of fresh figs, whereas dried figs were mostly well tolerated. In 60 patients with isolated Ficus benjamina sensitization, the reactivity rates to the same foods were 83-40-0-0-0-0%. None of 32 mugwort pollen-allergic patients reacted to Moraceae fruits. Rabbit anti-Mal d 1 and patient sera reacted to a 17 kDa band in all Moraceae extracts. IgE binding to these proteins was completely inhibited by birch pollen and rBet v 1. Mass spectrometry identified several peptides from the 17 kDa fig, mulberry and jackfruit allergen with respectively 60%, 56% and 76% homology to Bet v 1. Fig and other Moraceae fruits contain allergens homologous to Bet v 1 and represent clinically relevant birch pollen-associated foods.