Proteomic analysis of the major birch allergen Bet v 1 predicts allergenicity for 15 birch species

Pollen of the European and Asian white birch (Betula pendula and B. platyphylla) causes hay fever in humans. The allergenic potency of other birch species is largely unknown. To identify birch trees with a reduced allergenicity, we assessed the immunochemical characteristics of 15 species and two hybrids, representing four subgenera within the genus Betula, while focusing on the major pollen allergen Bet v 1. Antigenic and allergenic profiles of pollen extracts from these species were evaluated by SDS-PAGE and Western blot using pooled sera of birch-allergic individuals. Tryptic digests of the Bet v 1 bands were analyzed by LC-MS(E) to determine the abundance of various Bet v 1 isoforms. Bet v 1 was the most abundant pollen protein across all birch species. LC-MS(E) confirmed that pollen of all species contained a mixture of multiple Bet v 1 isoforms. Considerable differences in Bet v 1 isoform composition exist between birch species. However, isoforms that are predicted to have a high IgE-reactivity prevailed in pollen of all species. Immunoblotting confirmed that all pollen extracts were similar in immune-reactivity, implying that pollen of all birch species is likely to evoke strong allergic reactions.     

Pyr c 1, the major allergen from pear (Pyrus communis), is a new member of the Bet v 1 allergen family

Pear is known as an allergenic food involved in the ‘oral allergy syndrome’ which affects a high percentage of patients allergic to birch pollen. The aim of this study was to clone the major allergen of this fruit, to express it as bacterial recombinant protein and to study its allergenic properties in relation to homologous proteins and natural allergen extracts. The coding region of the cDNA was obtained by a PCR strategy, cloned, and the allergen was expressed as His-Tag fusion protein. The fusion peptide was removed by treatment with cyanogen bromide. Purified non-fusion protein was subjected to allergenicity testing by the enzyme allergosorbent test (EAST), Western blotting, competitive inhibition assays, and basophil histamine release. The deduced protein sequence shared a high degree of identity with other major allergens from fruits, nuts, vegetables, and pollen, and with a family of PR-10 pathogenesis related proteins. The recombinant (r) protein was recognised by specific IgE from sera of all pear-allergic patients (n=16) investigated in this study. Hence, the allergen was classified as a major allergen and named Pyr c 1. The IgE binding characteristics of rPyr c 1 appeared to be similar to the natural pear protein, as was demonstrated by EAST-inhibition and Western blot-inhibition experiments. Moreover, the biological activity of rPyr c 1 was equal to that of pear extract, as indicated by basophil histamine release in two patients allergic to pears. The related major allergens Bet v 1 from birch pollen and Mal d 1 from apple inhibited to a high degree the binding of IgE to Pyr c 1, whereas Api g 1 from celery, also belonging to this family, had little inhibitory effects, indicating epitope differences between Bet v 1-related food allergens. Unlimited amounts of pure rPyr c 1 are now available for studies on the structure and epitopes of pollen-related food allergens. Moreover, the allergen may serve as stable and standardised diagnostic material.     

Crystallization and preliminary x-ray investigation at 2.0 Å resolution of Bet v 1, a birch pollen protein causing IgE-mediated allergy

The 17 kDa protein from Betula verrucosa (White Birch) pollen, Bet v 1, is the clinically most important birch pollen allergen causing immediate Type I IgE-mediated allergy. The three-dimensional structure of Bet v 1 and its IgE-binding epitopes are at present not known. In addition, the biological function of Bet v 1 in birch pollen is not fully established. In this work, Bet v 1 has been expressed in Escherichia coli as a recombinant protein, purified and crystallized. The space group of recombinant Bet v 1 crystals is orthorhombic C2221 with unit cell parameters a = 32.13 Å, b = 74.22 Å, and c = 118.60 Å. There is one Bet v 1 molecule per asymmetric unit and the water content is 41%. Crystals diffract to 2.0 Å resolution and a complete native data set was collected from a single crystal using CuK_α X-rays from a rotating anode. © 1996 Wiley-Liss, Inc.     

Source of Bet v 1 loaded inhalable particles from birch revealed

 Allergenic proteins present in pollen grains, when inhaled, interact with the airways to cause an attack of asthma in susceptible humans. In one system, grass pollen grains rupture osmotically in rainfall, releasing allergen-containing inhalable particles into the atmosphere. In contrast, birch tree pollen grains do not rupture under these conditions, yet the major allergen, Bet v 1, has been detected in the atmosphere in inhalable particles of unknown origin. It is possible that Bet v 1 may diffuse from intact settled pollen grains and the allergenic material may again become airborne, interacting with settled fine particles from other sources prior to resuspension. This study investigates the mechanism for the release of birch pollen allergen-containing inhalable particles from pollen grains. We propose the hypothesis that (1) airborne birch pollen grains settle on nearby leaf surfaces; (2) then, following light rainfall, the grains germinate and, (3) later, pollen tubes burst, releasing inhalable particles carrying Bet v 1 into the atmospheric aerosol.   We used microscopic analyses of pollen behaviour following anther opening, a Burkard volumetric trap for pollen counts and a high volume air sampler with a two-stage cascade impactor for quantitative immunochemical analyses of Bet v 1. On dry days of high birch pollen count (48 grains/m³, 1.5 ng/m³ of Bet v 1), we found that the surfaces of birch leaves became coated with pollen. This ”pollen rain” is a source of secondary emission of allergens into the atmosphere. We observed that following light rainfall (<1 mm per day), about 80% of the birch pollen grains germinated, producing pollen tubes, especially in the sticky surface secretions of leaf glands. These pollen tubes may grow up to 300 μm in length prior to rupturing, each releasing about 400 starch granules coated with allergen molecules that may, after drying, be dispersed into the aerosol. On these days following light rainfall, the highest atmospheric levels of Bet v 1 (1.18 ng/m³) are associated with inhalable particles. Following heavy rainfall, both pollen and inhalable particles are washed from the atmosphere. Immunoprinting studies show that Bet v 1 is associated with starch granules rather than the smaller orbicules. Bet v 1 is present in the atmosphere in large particles, i.e. in particular pollen grains and in inhalable particles, i.e. in particular starch granules. Received: 28 May 1997 / Revision accepted: 18 August 1997     

Analysis of epitope-specific immune responses induced by vaccination with structurally folded and unfolded recombinant Bet v 1 allergen derivatives in man

Previously, we have constructed recombinant derivatives of the major birch pollen allergen, Bet v 1, with a more than 100-fold reduced ability to induce IgE-mediated allergic reactions. These derivatives differed from each other because the two recombinant Bet v 1 fragments represented unfolded molecules whereas the recombinant trimer resembled most of the structural fold of the Bet v 1 allergen. In this study, we analyzed the Ab (IgE, IgG subclass, IgA, IgM) response to Bet v 1, recombinant and synthetic Bet v 1-derived peptides in birch pollen allergic patients who had been vaccinated with the derivatives or adjuvant alone. Furthermore, we studied the induction of IgE-mediated skin responses in these patients using Bet v 1 and Bet v 1 fragments. Both types of vaccines induced a comparable IgG1 and IgG4 response against new sequential epitopes which overlap with the conformational IgE epitopes of Bet v 1. This response was 4- to 5-fold higher than that induced by immunotherapy with birch pollen extract. Trimer more than fragments induced also IgE responses against new epitopes and a transient increase in skin sensitivity to the fragments at the beginning of therapy. However, skin reactions to Bet v 1 tended to decrease one year after treatment in both actively treated groups. We demonstrate that vaccination with folded and unfolded recombinant allergen derivatives induces IgG Abs against new epitopes. These data may be important for the development of therapeutic as well as prophylactic vaccines based on recombinant allergens.     

Molecular investigations of pathogenesis-related Bet v 1 homologues in Passiflora (Passifloraceae)

The major birch pollen allergen, Bet v 1, responsible for allergic reactions in many areas of the world, is homologous to a large number of pathogenesis-related proteins (PRs), identified as PR10. As part of a long-range investigation of these types of proteins and of evolution in Passiflora,DNA sequences from eight Bet v 1 homologue isoforms were obtained from five species of this genus in Brazil, and their sequences compared among themselves and with 30 others from 8 different species, classified in different taxonomic units. The objective was a first characterization of these PRs in wild passionflowers, and their use for evolutionary and applied investigations. High interspecific, but low intraspecific variability was observed, as expected from multigenic families subjected to concerted evolution. The relationships obtained both within Passiflora and between it and seven other genera probably best reflect functional similarities than evolutionary history.     

Analysis of allergens in ambient aerosols: Comparison of areas subjected to different levels of air pollution

Recent studies describe interactions of pollen surfaces with aerosol particles; pollen surfaces undergo morphological changes and the release of allergens and allergenic fragments from the pollen can be enhanced. Thus allergens from pollen can be found in particle size fractions much smaller than undamaged pollen (<5 μm). This may explain allergic reactions in parts of the lungs which cannot be reached by undamaged pollen. In Switzerland the birch tree (betula verrucosa) major allergen Bet v 1 and the grass (phleum pratense) pollen major allergen Phl p 5 are of particular relevance for inducing pollinosis. In this study aerosols of different aerodynamic diameters were sampled by Andersen-Impactors over 18 months. Sampling areas are subjected to different levels of air pollution (Zürich, Switzerland, urban; Payerne, Switzerland, rural; Davos, Switzerland, alpine). Samples were scanned by electron microscopy and submitted to specific allergen assays (ELISA) for birch pollen major allergen Bet v 1 and grass pollen major allergen Phl p 5 respectively. Particle and major allergen concentrations were highest in Zürich, followed by Payerne and, significantly lower, Davos. Scanning electron microscopy investigations showed interactions of aerosols with pollen surfaces in Zürich and Payerne. The presence of Bet v 1 in smaller aerosol fractions was demonstrated in Zürich and Payerne some weeks before and after birch pollen was counted. An erratum to this article is available at .     

Analysis of allergens in ambient aerosols: Comparison of areas subjected to different levels of air pollution

Recent studies describe interactions of pollen surfaces with aerosol particles; pollen surfaces undergo morphological changes and the release of allergens and allergenic fragments from the pollen can be enhanced. Thus allergens from pollen can be found in particle size fractions much smaller than undamaged pollen (<5m). This may explain allergic reactions in parts of the lungs which cannot be reached by undamaged pollen. In Switzerland the birch tree (betula verrucosa) major allergen Bet v 1 and the grass (phleum pratense) pollen major allergen Phl p 5 are of particular relevance for inducing pollinosis. In this study aerosols of different aerodynamic diameters were sampled by Andersen-Impactors over 18 months. Sampling areas are subjected to different levels of air pollution (Zürich, Switzerland, urban; Payerne, Switzerland, rural: Davos, Switzerland, alpine). Samples were scanned by electron microscopy and submitted to specific allergen assays (ELISA) for birch pollen major allergen Bet v 1 and grass pollen major allergen Phl p 5 respectively. Particle and major allergen concentrations were highest in Zürich, followed by Payerne and, significantly lower, Davos. Scanning electron microscopy investigations showed interactions of aerosols with pollen surfaces in Zürich and Payerne. The presence of Bet v 1 in smaller aerosol fractions was demonstrated in Zürich and Payerne some weeks before and after birch pollen was counted.     

Bet v 1 proteins, the major birch pollen allergens and members of a family of conserved pathogenesis-related proteins, show ribonuclease activity in vitro

The Bet v. 1 gene family of birch encodes the major pollen allergens as well as pathogenesis-related (PR) proteins that are induced by microbes in somatic tissues. These PR proteins belong to a group of conserved intracellular defense-related proteins that have been termed 'ribonuclease-like' PR proteins, on the basis of the partial sequence homology observed between PR1, a Bet v 1-homologue from parsley, and a recently characterized ginseng ribonuclease. However, this enzymatic activity has not yet been demonstrated, not for any of the members of this family of PR proteins, nor for the related pollen allergens. We have investigated the possible nuclease activity of Bet v 1 using apparently homogeneous preparations of natural Bet v 1 purified from birch pollen, and a recombinant non-fusion protein purified from E. coli extracts. We report here that Bet v 1 proteins indeed possess an intrinsic ribonucleolytic activity as they can digest different RNA substrates in vitro, but show no activity on single or double-stranded DNA.     

Birch pollen-associated peanut allergies in children

Panallergens show structural similarities, and they are responsible for many cross-reactions between pollen and plant food sources. The aim of the present study was to investigate IgE reactivity to peanut allergen components in children with birch pollen allergy. Patients experienced symptoms of allergic asthma, allergic rhinitis, and urticaria, and they underwent a complete diagnostic evaluation, including skin prick test (SPT), specific IgE (sIgE) to birch pollen allergen (t3), peanut allergen (f13). In addition, measurement of sIgE to the major birch allergen components, Betula verrucosa (Bet v1, Bet v2), and to peanut allergen components, Arachis hypogaea (genuine componens: Ara h1, Ara h2, Ara h3, and cross-reactive Ara h8) was performed, by using a microarray technique (component resolved diagnosis, CRD). SPT to birch extract was positive in all children, and SPT to peanut extract was positive in 51 % of them. sIgE to both allergens was increased in 39 % of children, 55 % of them had increased sIgE (t3), and one child had increased sIgE (f13). CRD results confirmed that some children were sensitized to Bet v1 only, and some children to genuine Ara h only. Bet v1/Ara h8 cross-reactivity was found in 16 % of children. Results of the present study reveal that SPT, sIgE, and CRD may detect sensitization and co-sensitization with birch and peanut allergens/allergen components, and CRD may help to differentiate sensitization to genuine peanut components from sensitization to peanut cross-reactive component in birch-sensitive children. Diagnostic approach has to be individualized for each patient.     

Rapid production of the major birch pollen allergen Bet v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo characterization.

Type I allergies are immunological disorders that afflict a quarter of the world's population. Improved diagnosis of allergic diseases and the formulation of new therapeutic approaches are based on the use of recombinant allergens. We describe here for the first time the application of a rapid plant-based expression system for a plant-derived allergen and its immunological characterization. We expressed our model allergen Bet v 1, the major birch pollen allergen, in the tobacco-related species Nicotiana benthamiana using a tobacco mosaic virus vector. Two weeks postinoculation, plants infected with recombinant viral RNA containing the Bet v 1 coding sequence accumulated the allergen to levels of 200 microg/g leaf material. Total nonpurified protein extracts from plants were used for immunological characterizations. IgE immunoblots and ELISA (enzyme-linked immunoassay) inhibition assays showed comparable IgE binding properties for tobacco recombinant (r) Bet v 1 and natural (n) Bet v 1, suggesting that the B cell epitopes were preserved when the allergen was expressed in N. benthamiana plants. Using a murine model of type I allergy, mice immunized with crude leaf extracts containing Bet v 1 with purified rBet v 1 produced in E. coli or with birch pollen extract generated comparable allergen-specific IgE and IgG1 antibody responses and positive type I skin test reactions. These results demonstrate that nonpurified Bet v 1 overexpressed in N. benthamina has the same immunogenicity as purified Bet v 1 produced in E. coli or nBet v 1. We therefore conclude that this plant expression system offers a viable alternative to fermentation-based production of allergens in bacteria or yeasts. In addition, there may be a broad utility of this system for the development of new and low-cost vaccination strategies against allergy.     

The Influence of Recombinant Production on the Immunologic Behavior of Birch Pollen Isoallergens

Background

Allergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity.

Methodology

Therefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity.

Conclusion

The experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.     

Sequential and structural homology between intracellular pathogenesis-related proteins and a group of latex proteins

The intracellular pathogenesis-related proteins have been identified in a broad range of flowering plants. Some display quite different patterns of expression, in many cases unrelated to the pathogenic response. Nevertheless, these proteins are all very similar and in most cases share more than 35% sequence identity. In this report we investigate the significance of a rather weak similarity between the intracellular pathogenesis-related (IPR or PR-10) proteins and a group of proteins identified in the latex of opium poppy and in Arabidopsis, among others. A sequence analysis held together with the recently published three-dimensional structure of Bet v 1, an IPR protein from birch pollen, strongly suggests sequential and structural homology between the two protein families.     

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

Background  

Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MS^E was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.     

Molecular characterization of human IgG monoclonal antibodies specific for the major birch pollen allergen Bet v 1. Anti-allergen IgG can enhance the anaphylactic reaction

We report the molecular characterization of five human monoclonal antibodies, BAB1-5 (BAB1: IgG(1); BAB4: IgG(2); BAB2, 3, 5: IgG(4)), with specificity for the major birch pollen allergen, Bet v 1. BAB1-5 were obtained after immunotherapy and contained a high degree of somatic mutations indicative of an antigen-driven affinity maturation process. While BAB1 inhibited the binding of patients IgE to Bet v 1, BAB2 increased IgE recognition of Bet v 1, and, even as Escherichia coli-expressed Fab, augmented Bet v 1-induced immediate type skin reactions. The demonstration that IgG antibodies can enhance allergen-induced allergic reactions is likely to explain the unpredictability of specific immunotherapy.     

Genetic engineering of a hypoallergenic trimer of the major birch pollen allergen Bet v 1.

An estimated 100 million individuals suffer from birch pollen allergy. Specific immunotherapy, the only curative allergy treatment, can cause life-threatening anaphylactic side effects. Here, we report the genetic engineering of a recombinant trimer consisting of three covalently linked copies of the major birch pollen allergen, Bet v 1. The trimer exhibited profoundly reduced allergenic activity but contained similar secondary structures such as Bet v 1 wild type, Bet v 1-specific B cell and T-cell epitopes, and induced Th1 cytokine release. As immunogen, rBet v 1 trimer induced IgG antibodies, which blocked patients' IgE binding to Bet v 1 and related allergens. Thus, rBet v 1 trimer represents a novel hypoallergenic vaccine prototype for treatment of one of the most frequent allergy forms.     

Functional analysis of birch pollen allergen Bet v 1-specific regulatory T cells

Allergen-specific immunotherapy using peptides is an efficient treatment for allergic diseases. Recent studies suggest that the induction of CD4+ regulatory T (Treg) cells might be associated with the suppression of allergic responses in patients after allergen-specific immunotherapy. Our aim was to identify MHC class II promiscuous T cell epitopes for the birch pollen allergen Bet v 1 capable of stimulating Treg cells with the purpose of inhibiting allergic responses. Ag-reactive CD4+ T cell clones were generated from patients with birch pollen allergy and healthy volunteers by in vitro vaccination of PBMC using Bet v 1 synthetic peptides. Several CD4+ T cell clones were induced by using 2 synthetic peptides (Bet v 1(141-156) and Bet v 1(51-68)). Peptide-reactive CD4+ T cells recognized recombinant Bet v 1 protein, indicating that these peptides are produced by the MHC class II Ag processing pathway. Peptide Bet v 1(141-156) appears to be a highly MHC promiscuous epitope since T cell responses restricted by numerous MHC class II molecules (DR4, DR9, DR11, DR15, and DR53) were observed. Two of these clones functioned as typical Treg cells (expressed CD25, GITR, and Foxp3 and suppressed the proliferation and IL-2 secretion of other CD4+ T cells). Notably, the suppressive activity of these Treg cells required cell-cell contact and was not mediated through soluble IL-10 or TGF-beta. The identified promiscuous MHC class II epitope capable of inducing suppressive Treg responses may have important implication for the development of peptide-based Ag-specific immunotherapy to birch pollen allergy.     

Quality Control of Biomedicinal Allergen Products – Highly Complex Isoallergen Composition Challenges Standard MS Database Search and Requires Manual Data Analyses

Allergy against birch pollen is among the most common causes of spring pollinosis in Europe and is diagnosed and treated using extracts from natural sources. Quality control is crucial for safe and effective diagnosis and treatment. However, current methods are very difficult to standardize and do not address individual allergen or isoallergen composition. MS provides information regarding selected proteins or the entire proteome and could overcome the aforementioned limitations. We studied the proteome of birch pollen, focusing on allergens and isoallergens, to clarify which of the 93 published sequence variants of the major allergen, Bet v 1, are expressed as proteins within one source material in parallel. The unexpectedly complex Bet v 1 isoallergen composition required manual data interpretation and a specific design of databases, as current database search engines fail to unambiguously assign spectra to highly homologous, partially identical proteins. We identified 47 non-allergenic proteins and all 5 known birch pollen allergens, and unambiguously proved the existence of 18 Bet v 1 isoallergens and variants by manual data analysis. This highly complex isoallergen composition raises questions whether isoallergens can be ignored or must be included for the quality control of allergen products, and which data analysis strategies are to be applied.     

Cross-reactivity of pollen and food allergens: soybean Gly m 4 is a member of the Bet v 1 superfamily and closely resembles yellow lupine proteins

In many cases, patients allergic to birch pollen also show allergic reactions after ingestion of certain fruits or vegetables. This observation is explained at the molecular level by cross-reactivity of IgE antibodies induced by sensitization to the major birch pollen allergen Bet v 1 with homologous food allergens. As IgE antibodies recognize conformational epitopes, a precise structural characterization of the allergens involved is necessary to understand cross-reactivity and thus to develop new methods of allergen-specific immunotherapy for allergic patients. Here, we report the three-dimensional solution structure of the soybean allergen Gly m 4, a member of the superfamily of Bet v 1 homologous proteins and a cross-reactant with IgE antibodies originally raised against Bet v 1 as shown by immunoblot inhibition and histamine release assays. Although the overall fold of Gly m 4 is very similar to that of Bet v 1, the three-dimensional structures of these proteins differ in detail. The Gly m 4 local structures that display those differences are also found in proteins from yellow lupine with known physiological function. The three-dimensional structure of Gly m 4 may thus shed some light on the physiological function of this subgroup of PR10 proteins (class 10 of pathogenesis-related proteins) and, in combination with immunological data, allow us to propose surface patches that might represent cross-reactive epitopes.