Different IgE reactivity profiles in birch pollen-sensitive patients from six European populations revealed by recombinant allergens: an imprint of local sensitization

BACKGROUND: Sensitivity to birch pollen allergens is a common feature among European patients with seasonal pollen allergy. In this in vitro study, we examined the specific serum IgE binding profiles to individual birch pollen allergens in birch-sensitive patients from six European populations. METHODS: The study included 242 patients from Finland, Sweden, Austria, France, Switzerland and Italy. All suffered from seasonal rhinoconjunctivitis and/or asthma. Their sera were analyzed for specific IgE reactivity to individual birch pollen allergens (recombinant Bet v 1, Bet v 2 and Bet v 4) and natural birch pollen extract using Pharmacia CAP System and immunoblotting. RESULTS: Almost all Finnish, Swedish and Austrian sera contained IgE specific for Bet v 1 (>or=98%). Bet v 1-specific IgE antibodies were found in 90% of the French sera, and in 65 and 62% of the sera from Switzerland and Italy, respectively. Few Finnish (2%) and Swedish (12%) patients had IgE to Bet v 2, while Bet v 2 reactivity was more common in the other populations (20-43%). Reactivity to Bet v 4 was rare in all populations (5-11%) except for the Italian patients, in whom 3 of 11 sera were positive (27%). The immunoblot results supported the specific IgE profiles obtained with Pharmacia CAP System showing a broader IgE reactivity profile in patients from central and southern Europe as compared to northern Europe. CONCLUSION: Component-resolved allergy diagnosis with recombinant allergens reveals that the IgE reactivity profiles to individual birch pollen allergens vary between European populations. This observation may be explained by sensitization to different allergen sources and will have an impact on allergen-specific prevention and therapy strategies.     

The importance of recombinant allergens for diagnosis and therapy of IgE-mediated allergies

In the past 10 years, a considerable number of cDNAs coding for allergens have been isolated and expressed. Intensive investigations showed that recombinant allergens and their respective natural counterparts possess comparable properties with respect to structure, function and interaction with the immune system. Recent studies documented that in vitro as well as in vivo diagnosis of IgE-mediated allergic diseases can be successfully improved by the application of recombinant allergens. In addition, new strategies for a safer specific immunotherapy (SIT) have been developed based on the knowledge of the primary structures of allergens. Naturally occurring isoforms of allergens as well as recombinant allergens with modified amino acid sequences show very low IgE binding capacity but strong T cell-stimulatory activity and represent possible candidates. In case of Bet v 1, the major birch pollen allergen, isoforms d, g and l and a Bet v 1a mutant, produced by site-directed mutagenesis resulting in 6 amino acid exchanges, fulfilled the above mentioned criteria. In a third approach, two adjacent peptides covering the entire Bet v 1a sequence were produced in an Escherichia coli expression system. These peptides contained most of the relevant T cell epitopes, but lost their IgE binding capacity and, thus, their ability to activate mast cells and basophils of sensitized patients. Our results suggest that allergen variants (isoforms, mutants, T cell epitope-containing peptides) may be used as 'hypoallergenic agents' in SIT.     

The allergen profile of beech and oak pollen

Background Beech and oak pollen are potential allergen sources with a world‐wide distribution. Objective We aimed to characterize the allergen profile of beech and oak pollen and to study cross‐reactivities with birch and grass pollen allergens. Methods Sera from tree pollen‐allergic patients with evidence for beech and oak pollen sensitization from Basel, Switzerland, (n=23) and sera from birch pollen‐allergic patients from Vienna, Austria, (n=26) were compared in immunoblot experiments for IgE reactivity to birch (Betula pendula syn. verrucosa), beech (Fagus sylvatica) and oak (Quercus alba) pollen allergens. Subsequently, beech and oak pollen allergens were characterized by IgE inhibition experiments with purified recombinant and natural allergens and with allergen‐specific antibody probes. Birch‐, beech‐ and oak pollen‐specific IgE levels were determined by ELISA. Results Beech and oak pollen contain allergens that cross‐react with the birch pollen allergens Bet v 1, Bet v 2 and Bet v 4 and with the berberine bridge enzyme‐like allergen Phl p 4 from timothy grass pollen. Sera from Swiss and Austrian patients exhibited similar IgE reactivity profiles to birch, beech and oak pollen extracts. IgE levels to beech and oak pollen allergens were lower than those to birch pollen allergens. Conclusion IgE reactivity to beech pollen is mainly due to cross‐reactivity with birch pollen allergens, and a Phl p 4‐like molecule represented another predominant IgE‐reactive structure in oak pollen. The characterization of beech and oak pollen allergens and their cross‐reactivity is important for the diagnosis and treatment of beech and oak pollen allergy.     

Detection of specific IgE antibodies in the sera of patients allergic to birch pollen using recombinant allergens Bet v 1, Bet v 2, Bet v 4: evaluation of different IgE reactivity profiles

BACKGROUND: Birch pollen is a significant cause of immediate hypersensitivity among susceptible subjects in temperate climates, affecting 5-54% of the population in western Europe. We examined the specific serum IgE antibodies towards recombinant allergens Bet v 1, Bet v 2 and Bet v 4 in birch-sensitive patients from the province of Cuneo, north-west Italy. METHODS: Sera were obtained from 372 patients with symptomatic birch pollen-induced allergic rhinitis and/or asthma. A subgroup of these patients suffered from oral allergy syndrome after eating apple. Their sera were evaluated for specific IgE against natural birch pollen and apple extract, as well as Bet v 1, Bet v 2 and Bet v 4 using Pharmacia CAP system (Pharmacia, Uppsala, Sweden). RESULTS: Of 372 patients 215 (57.80%) had serum-specific IgE towards Bet v 1. A total of 166 sera (44.62%) contained serum-specific IgE to Bet v 2, while Bet v 4 IgE reactivity was documented in 35 subjects (9.41%). Moreover, 146 (39.25%) patients were monosensitized to Bet v 1; 96 (25.81%) patients were monosensitized to Bet v 2; only four sera (1.08%) contained specific IgE towards Bet v 4. Thirty-nine sera (11.02%) did not contain specific IgE to these individual birch pollen allergens. Of course, all 372 sera (100%) had specific IgE against natural birch pollen extract, of which 162 (43.55%) contained specific IgE to apple extract (75.35% of Bet v 1 positive sera). CONCLUSION: In this study we observed that three birch pollen recombinant allergens alone, could sufficiently identify 90% of birch pollen-sensitive patients. Therefore, for a more precise IgE profile of patients allergic to birch, further purified birch pollen allergens (i.e. Bet v 6, Bet v 7, Bet v 8) will be required.     

Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollen allergen

Background Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffer froin allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis. leading to death.
Objective The aim of this study was to evaluate recombinant Bet v I, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy.
Methods A group of 51 birch pollen allergic patients and eight non-allergic control individuals were tested for birch pollen allergy by skin-prick and intradennal testing, comparing commercial birch pollen extracts with recombinant Bet v I. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting.
Results Recombinant Bet v I allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained in any in vitro assay system (ELISA. Westernblot) or by skin testing (skin-prick, intradermal test) with recombinant Bet v I.
Conclusion Our results document that recombinant Bet v I produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in > 95% of birch pollen allergic patients.     

Bet v 1142-156 is the dominant T-cell epitope of the major birch pollen allergen and important for cross-reactivity with Bet v 1-related food allergens

BACKGROUND: Individuals with birch pollen allergy frequently experience hypersensitivity reactions to certain foods, primarily because of IgE antibodies specific for the major birch pollen allergen Bet v 1 that cross-react with homologous food allergens. OBJECTIVE: We sought to characterize the major T-cell epitopes of Bet v 1 and to investigate their involvement in the cellular cross-reactivity with homologous food allergens. METHODS: T-cell epitope mapping of Bet v 1 was performed by testing Bet v 1-specific T-cell lines derived from 57 individuals with birch pollen allergy, with overlapping peptides representing the entire allergen. T-cell lines and T-cell clones were stimulated with Bet v 1-related major allergens from apple (Mal d 1), cherry (Pru av 1), hazelnut (Cor a 1), celery (Api g 1), carrot (Dau c 1), and soybean (Gly m 4) and with peptides deduced from the C-terminal amino acid sequences of these molecules. Results Bet v 1 142-156 , positioned in the highly conserved C-terminal region of Bet v 1, was identified as the major T-cell epitope recognized by 61% of individuals. Most T lymphocytes specific for Bet v 1 142-156 were activated by one or more homologous food proteins or the respective peptides, as indicated by proliferation and cytokine production. CONCLUSION: The major T-cell epitope of Bet v 1, Bet v 1 142-156 , plays an important role in the cellular cross-reactivity between this respiratory allergen and related food allergens. Thus T lymphocytes specific for Bet v 1 142-156 might be activated by various Bet v 1-related food allergens in vivo, even out of the pollen season.     

Characterization of api g 1.0201, a new member of the Api g 1 family of celery allergens

BACKGROUND: The association of pollinosis with allergy to plant foods occurs in up to 70% of tree pollen-allergic patients. In recent years, some of the relevant cross-reacting proteins have been characterized at the molecular and immunological level. Api g 1 has been identified as the celery homologue of the major birch pollen allergen, Bet v 1. Although a number of Bet v 1 isoforms have been characterized from birch pollen, little is known about isoforms of food allergens and their allergenic features. METHODS: Api g 1.0201, an isoform of Api g 1, was isolated from a cDNA library, cloned and sequenced. The cDNA was expressed in Escherichia coli and the purified recombinant protein was tested in immunoblots. RESULTS: Api g 1.0201 displays 72% sequence similarity to the previously identified Api g 1.0101 and consists of 159 amino acid residues. The sequence of Api g 1.0201 has five additional amino acid residues at the carboxy-terminus as compared to Api g 1.0101. Purified recombinant Api g 1.0201 is recognized by IgE from the sera of celery-allergic patients, as well as by the murine monoclonal anti-Bet v 1 antibody. In general, this isoform displays a weaker IgE-binding capacity than Api g 1.0101, as concluded from immunoblotting experiments. Results from inhibition assays revealed that IgE-binding to Api g 1.0201 is only slightly reduced by preincubation with either purified recombinant Api g 1.0101 or purified recombinant Bet v 1a. Total inhibition was only achieved when using purified natural Bet v 1. CONCLUSIONS: At present, little is known about the IgE-binding capacity of isoforms of Bet v 1 homologues of food allergens. Identification and characterization of such isoforms may help to contribute to a better understanding of food allergy and the observed cross-reactivity to pollen allergy.     

Soy allergy in perspective

PURPOSE OF REVIEW: The purpose of this paper is to review and discuss studies on soy allergy. RECENT FINDINGS: In Central Europe soy is a clinically relevant birch pollen-related allergenic food. Crossreaction is mediated by a Bet v 1 homologous protein, Gly m 4. Additionally, birch pollen allergic patients might acquire through Bet v 1 sensitization allergies to mungbean or peanut, in which Vig r 1 and Ara h 8 are the main cross-reactive allergens. Threshold doses in soy allergic individuals range from 10 mg to 50 g of soy and are more than one order of magnitude higher than in peanut allergy. No evidence was found for increased allergenicity of genetically modified soybeans. SUMMARY: In Europe, both primary and pollen-related food allergy exist. The diagnosis of legume allergy in birch pollen-sensitized patients should not be excluded on a negative IgE testing to legume extracts. Bet v 1 related allergens are often underrepresented in extracts. Gly m 4 from soy and Ara h 8 from peanut are nowadays commercially available and are recommended in birch pollen allergic patients with suspicion of soy or peanut allergy, but negative extract-based diagnostic tests to screen for IgE specific to these recombinant allergens.     

Immunologic significance of respirable atmospheric starch granules containing major birch allergen Bet v 1

BACKGROUND: Birch-pollen allergens are an important cause of early spring hay fever and allergic asthma. Recently, we reported a mechanism for the release of respirable allergenic particles from birch pollen containing the major allergen Bet v 1. In this study, we aimed to assess the immunologic significance of the released Bet v 1-containing starch granules in the environment. METHODS: A two-site monoclonal antibody-based assay (ELISA) was employed to quantitate Bet v 1 in high-volume air sampler filter extracts, and immunogold-labelling was used on sections of these extracts to localize Bet v 1. Immunoblot analyses were performed with pooled sera from patients sensitive to birch pollen. RESULTS: Atmospheric starch granules contained Bet v 1, and the concentration increased upon light rainfall. Sera from patients allergic to birch allergens recognized extracts from isolated starch granules. CONCLUSIONS: The clinical implications of these findings are that starch granules released from birch pollen are potentially able to trigger allergic asthmatic reactions to Bet v 1, since the allergen occurs in respirable particles. Thus, clinicians can advise asthma patients to remain indoors on days of light rainfall during the birch-pollen season to avoid high levels of allergen exposure.     

Bet v 1, the major birch pollen allergen, initiates sensitization to Api g 1, the major allergen in celery: evidence at the T cell level

Due to IgE cross-reactivity, birch pollen-allergic individuals frequently develop type I hypersensitivity reactions to celery tuber. We evaluated the T cell response to the major allergen in celeriac, Api g 1, and the cellular cross-reactivity with its homologous major allergen in birch pollen, Bet v 1. Api g 1-specific T cell lines (TCL) and clones (TCC) were established from peripheral blood mononuclear cells of allergic patients. Epitope mapping of Api g 1 with overlapping Api g 1-derived peptides revealed one dominant T cell-activating region, Api g 1(109-126). TCL and TCC generated with Api g 1 cross-reacted with the birch pollen allergen and, although initially stimulated with the food allergen, cellular responses to Bet v 1 were stronger than to Api g 1. Epitope mapping with Bet v 1-derived peptides revealed that T cells specific for several distinct epitopes distributed over the complete Bet v 1 molecule could be activated by Api g 1. Bet v 1(109-126) was identified as the most important T cell epitope for cross-reactivity with Api g 1. This epitope shares 72% amino acid sequence similarity with the major T cell-activating region of the food allergen, Api g 1(109-126). Our data provide evidence that humoral as well as cellular reactivity to the major celery allergen is predominantly based on cross-reactivity with the major birch pollen allergen. The activation of Bet v 1-specific Th2 cells by Api g 1, in particular outside the pollen season, may have consequences for birch pollen-allergic individuals.     

Birch pollen-related food allergy to legumes: identification and characterization of the Bet v 1 homologue in mungbean (Vigna radiata), Vig r 1

BACKGROUND: Recently allergic reactions to legumes mediated by Bet v 1-homologous food allergens were described for soy and peanut. In this study we assessed allergic reactions to another legume, to mungbean seedlings, and identified its Bet v 1-homologous allergen Vig r 1. METHODS: Ten patients were selected who had a history of allergic reactions to mungbean seedlings and a respiratory allergy to birch pollen. The Bet v 1 homologue in mungbean seedlings, Vig r 1, was cloned by a PCR strategy, expressed in Escherichia coli, and purified by preparative SDS-PAGE. In all sera, specific IgE against birch pollen, Bet v 1, Bet v 2, Vig r 1, and the Bet v 1 homologues in soy (Gly m 4) and cherry (Pru av 1) was determined by CAP-FEIA. Cross-reactivity of specific IgE with Vig r 1, Bet v 1, Gly m 4, and Pru av 1 was assessed by immunoblot inhibition. Expression of Vig r 1 during development of mungbean seedlings and under wounding stress was analysed by immunoblotting. The Vig r 1 double band was analysed by matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: All patients were sensitized to birch pollen and Bet v 1, 20% to Bet v 2, and 90% to Gly m 4. Seventy percent of the patients showed IgE binding to a double band at 15 kDa in mungbean extract that was inhibited after pre-incubation of sera with rBet v 1. PCR cloning revealed that the mungbean homologue of Bet v 1 had a molecular weight of 16.2 kDa, a calculated pI of 4.6% and 42.8% amino acid sequence identity with Bet v 1. MS analysis confirmed similarity of the double band with the deduced Vig r 1 sequence, but also indicated the existence of other Vig r 1 isoforms. ImmunoCAP analysis detected IgE against Vig r 1 in 80% of the sera. IgE binding to Vig r 1 was inhibited with Gly m 4 in six of six and with rPru av 1 in four of six patients. Vig r 1 expression occurred during development of seedlings and was increased by wounding stress. CONCLUSIONS: Food allergy to mungbean seedlings can be caused by primary sensitization to birch pollen and is mediated by Vig r 1 in the majority of the patients with birch pollen-related allergy to mungbean seedlings.     

Molecular basis of allergic cross-reactivity between group 1 major allergens from birch and apple.

Patients allergic to birch pollen often also react with fruits and vegetables, such as apple. The major cause of cross-reactivity between birch and apple is biochemical and immunological similarity between the major allergens, Bet v 1 and Mal d 1, as demonstrated by serological and cellular immunoassays. In addition, birch pollen-specific therapeutic allergy vaccination has been shown to improve allergic symptoms caused by oral ingestion of apple. Detailed analysis of molecular surface areas based on the crystal structure of Bet v 1, and primary sequence alignment, identify potential epitopes for cross-reactive antibodies. Two or more conserved patches are identified when comparing Bet v 1 and Mal d 1, thus providing a molecular model for serological cross-reactivity involving more than one IgE-binding epitope. A minimum of two epitopes would be necessary for cross-linking of receptor bound IgE in functional histamine release assays and skin test. Individual amino acid substitutions, as occurring in isoallergenic variation, may, however, have a dramatic effect on epitope integrity if critical residues are affected. Thus, one area large enough to accommodate antibody-binding epitopes shared by all known Mal d 1 isoallergens and variants is identified, as well as areas shared by Bet v 1 and individual Mal d 1 isoallergens or variants. The occurrence of limited epitope coincidence between Bet v 1 and Mal d 1 is in agreement with the observation that some, but not all, birch pollen allergic patients react with apple, and that the epitope repertoire recognised by the IgE of the individual patients determines the degree of cross-reactivity.     

Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins.

BACKGROUND: Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods. OBJECTIVE: The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties. METHODS: On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2. RESULTS: Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy. In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1. CONCLUSIONS: This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy. Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions.     

Fruit-pollen-latex cross-reactivity: implication of profilin (Bet v 2).

BACKGROUND: An association between allergy to fruits and latex, and between pollen and plant-derived food has been described. The cross-reactive structures responsible for these associations have not yet been completely elucidated. METHODS: IgE reactivity to the recombinant allergens Bet v 1 and Bet v 2, different pollens, natural latex, papain, and bromelain was investigated in 29 patients with allergy to fruits or vegetables who lived in an area without birch trees. RESULTS: Exactly 79.3% of patients were allergic to grass pollen, and two of them had clinical allergy to latex. Serum IgE reactivity (CAP) to birch pollen was found in 65% of patients, to Bet v 2 in 51.7%, to Bet v 1 in 3.4%, to latex in 58.6%, to bromelain in 51.7%, and to papain in 17.2% of patients. All subjects with positive IgE to Bet v 2 had also reactivity to latex, grass, olive tree, birch, and mugwort pollens. The six patients not allergic to pollen did not show IgE reactivity to latex, Bet v 1, or Bet v 2. A significant correlation was found between CAP to latex with Bet v 2 (r=0.86, P<0.001), with birch (r=0.86, P<0.001), and with ryegrass (r=0.81, P<0.001). Immunoblotting using nine sera with positive CAP to birch pollen showed IgE-binding to a 15-kDa band that was recognized by antiprofilin monoclonal antibody. Bet v 2 CAP could be inhibited up to 52% by ryegrass and up to 23% by mugwort. CAP to latex was almost completely inhibited by ryegrass pollen with sera from five subjects without symptoms due to latex, whereas no inhibition was observed with serum from one patient with allergy to latex. CONCLUSIONS: Patients with allergy to plant-derived food and associated pollinosis showed a high frequency of IgE reactivity to Bet v 2, which may cause positive serum IgE determinations to latex and birch pollen due to the presence of cross-reactive epitopes. IgE reactivity to Bet v 2 may serve as an indicator of broad sensitization.     

Fagales pollen sensitization in a birch-free area: a respiratory cohort survey using Fagales pollen extracts and birch recombinant allergens (rBet v 1, rBet v 2, rBet v 4)

BACKGROUND: Birch allergy is one of the most common pollinosis in areas where exposure to high levels of birch pollen is common. Little is known about birch sensitivity in areas without birch pollen exposure and reactivity to birch-related species within the Fagales order. OBJECTIVE: the aim was to evaluate Fagales reactivity within a population not exposed to birch pollen using epidemiological, diagnostic, and laboratory approaches by means of extracts and allergenic molecules. METHODS: A cohort of 5335 respiratory allergic patients was screened by means of skin testing birch, hazel, and oak pollen extracts. Patients were from a birch-free area, but exposed to other Fagales pollen species. A subset of patients was from an intensively cultivated hazel area. A sample of the Fagales allergic population was tested with other Fagales pollen extract (alder, hornbeam, beech, chestnut) and with apple and hazelnut. IgE detection was performed with birch, hazel, oak, apple, and hazelnut extracts, and with Bet v 1, Bet v 2, Bet v 4, and bromelain. IgE immunoblots were performed using birch and hazel extracts. Epidemiological, clinical, and laboratory data were analysed by stratifying the allergic population. RESULTS: Twenty-five percent of the pollen allergic cohort was skin test positive to at least one of the three Fagales species. Combined reactivity to the three species was recorded in 80% of this cohort. Isolated hazel pollen reactivity was recorded in 13.5% of the Fagales allergic patients. Sixty-six percent of these subjects were from the intensively cultivated hazel area. Reactivity to apple and hazelnut was detected by skin test (40%) and IgE reactivity (60%), but only 19% of the positive patients reported symptoms related to at least one of the two foods. Reactivity to Bet v 1 was recorded in 84% of the birch/hazel/oak co-reactivity group, and in 28% of the subjects with the same co-reactivity, but associating a multiple pollen sensitization. IgE to Bet v 2 (50%) and Bet v 4 (23%) panallergens were recorded positive in the latter subset. Bet v 1 prevalence ranged between 48% and 21% among subgroups of patients coming from different areas. Furthermore, an IgE reactivity to hazel-restricted allergenic components was detected among subjects coming from the same area and having a hazel isolated reactivity. CONCLUSION: Fagales allergy can be found in birch-free areas caused by the exposure to other Fagales species. Birch allergens can be useful for mimicking the allergenic extract, but are also the exclusive tools for a fine diagnostic and epidemiological approach to Fagales pollen allergy. Allergenic molecules from the hazel family will increase the panel of available reagents for the molecule-based approach to allergy diagnosis and therapy.     

Skin test evaluation of genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1: results obtained with a mix of two recombinant Bet v 1 fragments and recombinant Bet v 1 trimer in a Swedish population before the birch pollen season.

BACKGROUND: More than 95% of birch pollen-allergic subjects react with the major birch pollen allergen, Bet v 1, and almost 60% of them are sensitized exclusively to this allergen. OBJECTIVE: The aim of this study was to compare the in vivo biologic activity of genetically engineered hypoallergenic derivatives of Bet v 1 (an equimolar mixture of 2 recombinant [r] Bet v 1 fragments and of rBet v 1 trimer) with that of rBet v 1 wild-type by skin prick and intradermal testing. METHODS: Birch pollen-allergic patients who had not received immunotherapy (n = 23), a group of allergic patients without birch pollen allergy (n = 12), and nonatopic persons (n = 8) from northern Europe (Sweden) underwent skin prick and intradermal testing with different concentrations of the recombinant allergens and commercial birch pollen extract before the birch pollen season. Immediate and late-phase reactions were recorded and allergen-specific IgE and IgG subclass responses were determined by CAP radioallergosorbent test and ELISA, respectively. RESULTS: Atopic persons without birch pollen allergy and nonatopic individuals did not have skin reactions to rBet v 1 wild-type and genetically engineered hypoallergenic derivatives. By intradermal testing, 8 of 23 and 13 of 23 birch pollen-allergic patients did not react with the highest concentration (1 microg/mL) of the rBet v 1 fragment mix and rBet v 1 trimer, respectively, compared with 1 with rBet v 1 wild type. Likewise, the highest concentration (100 microg/mL) of fragment mix or trimer failed to elicit a positive skin prick test in 18 of 23 and 15 of 23 patients in comparison with 0/23 with the monomer. No late reactions were observed. CONCLUSION: The recombinant hypoallergenic birch pollen allergens can probably be used for patient-tailored immunotherapy with a reduced risk to induce anaphylactic reactions.     

Characterization of the protective and therapeutic efficiency of a DNA vaccine encoding the major birch pollen allergen Bet v 1a

BACKGROUND: An estimated 100 million individuals suffer from birch pollen allergy. More than 95% of birch pollen-allergic subjects react with the major birch pollen allergen Bet v 1a, and almost 60% of them are sensitized exclusively to this allergen. OBJECTIVE: DNA immunization using the Bet v 1a gene was evaluated with respect to its prophylactic and therapeutic efficacy. METHODS: A DNA vaccine containing the entire Bet v 1a cDNA under the control of a CMV-promoter was constructed. In order to estimate the protective efficiency, animals received three injections of this vaccine prior to sensitization with recombinant Bet v 1a. Vice versa, in a therapeutic approach, sensitization was followed by treatment with the DNA vaccine. RESULTS: The Bet v 1a DNA vaccine induced strong Bet v 1-specific antibody responses with a Th1-biased response type. Animals which received the DNA vaccine were protected against a following allergic sensitization with Bet v 1a. The protective effect was characterized by suppression of Bet v 1-specific immunoglobulin (Ig)E production, lack of basophil activation and enhanced interferon (IFN)-gamma expression. In a therapeutic situation, treatment of sensitized animals with DNA vaccines decreased IgE production, IgE-mediated basophil release and drastically reduced anaphylactic activity as measured by passive cutaneous anaphylaxis assays. Concerning the cellular immune response, DNA immunization induced a sustaining and dominant shift from a Th2 type response towards a balanced Th1/Th2 type response as indicated by increased IFN-gamma but unchanged IL-5 levels in lymphoproliferation assays. CONCLUSION: The results demonstrate the allergen-specific protective and therapeutic efficacy of a DNA vaccine encoding the clinically highly relevant allergen Bet v 1a indicating the suitability of this concept for the treatment of allergic diseases.     

Gastrointestinal digestion of Bet v 1-homologous food allergens destroys their mediator-releasing, but not T cell-activating, capacity

BACKGROUND: Food allergy to apples, hazelnuts, and celery is frequent in individuals with birch pollen allergy because IgE antibodies specific for the major birch pollen allergen, Bet v 1, cross-react with structurally related allergens in these foods. In addition, T lymphocytes specific for Bet v 1 also cross-react with these dietary proteins. OBJECTIVE: We sought to evaluate the effects of simulated gastrointestinal degradation of Bet v 1-related food allergens on their mediator-releasing and T cell-activating capacity. METHODS: Recombinant Mal d 1, Cor a 1.04, and Api g 1 were incubated separately with pepsin and trypsin. Binding of IgE was tested in immunoblots. After successive incubation with both enzymes, allergens were tested in mast cell mediator release assays and used to stimulate PBMCs and Bet v 1-specific T-cell lines and clones. Proteolytic fragments of allergens were analyzed and sequenced by means of mass spectrometry. RESULTS: Pepsin completely destroyed IgE binding of all allergens within 1 second, and trypsin completely destroyed IgE binding of all allergens within 15 minutes, except for the major hazelnut allergen, which remained intact for 2 hours of trypsinolysis. Allergens after gastrointestinal digestion did not induce basophil activation but induced proliferation in PBMCs from allergic and nonallergic individuals. Digested Mal d 1 and Cor a 1.04 still activated Bet v 1-specific T cells, whereas digested Api g 1 did not. Different proteolytic fragments of Mal d 1 and Cor a 1.04 matching relevant Bet v 1 T-cell epitopes were found. CONCLUSION: Gastrointestinal degradation of Bet v 1-related food allergens destroys their histamine-releasing, but not T cell-activating, property. Our data emphasize that birch pollen-related foods are relevant activators of pollen-specific T cells.