Birch Pollen Allergen Research Articles

Bet v 1 from Birch Pollen Is a Lipocalin-like Protein Acting as Allergen Only When Devoid of Iron by Promoting Th2 Lymphocytes

It is hypothesized that allergens are at the borderline of self and non-self and, through as yet elusive circumstances, mount a Th2 response for allergic sensitization. The major birch pollen allergen Bet v 1 is considered the prototype for the PR-10 protein family causing respiratory allergy. Here, we give structural evidence that Bet v 1 is a lipocalin-like protein with a striking structural resemblance to human lipocalin 2. Lipocalin 2 is highly expressed in the lung where it exerts immunoregulatory functions dependent on being loaded with siderophore-bound iron (holo-form) or not (apo-form). We demonstrate that similar to lipocalin 2, Bet v 1 is capable of binding iron via catechol-based siderophores. Thereby, calculated Kd values of 66 nm surpassed affinities to known ligands nearly by a power of 10. Moreover, we give functional evidence of the immunomodulatory capacity of Bet v 1 being dependent on its iron-loaded state. When incubated to human immune cells, only the apo-form of Bet v 1, but not the holo-form, was able to promote Th2 cells secreting IL13. These results provide for the first time a functional understanding on the allergenicity of Bet v 1 and a basis for future allergen immunotherapies counteracting Th2 immune responses on a molecular basis.

Do lipids influence the allergic sensitization process?

Allergic sensitization is a multifactorial process that is not only influenced by the allergen and its biological function per se but also by other small molecular compounds, such as lipids, that are directly bound as ligands by the allergen or are present in the allergen source. Several members of major allergen families bind lipid ligands through hydrophobic cavities or electrostatic or hydrophobic interactions. These allergens include certain seed storage proteins, Bet v 1–like and nonspecific lipid transfer proteins from pollens and fruits, certain inhalant allergens from house dust mites and cockroaches, and lipocalins. Lipids from the pollen coat and furry animals and the so-called pollen-associated lipid mediators are codelivered with the allergens and can modulate the immune responses of predisposed subjects by interacting with the innate immune system and invariant natural killer T cells. In addition, lipids originating from bacterial members of the pollen microbiome contribute to the outcome of the sensitization process. Dietary lipids act as adjuvants and might skew the immune response toward a TH2-dominated phenotype. In addition, the association with lipids protects food allergens from gastrointestinal degradation and facilitates their uptake by intestinal cells. These findings will have a major influence on how allergic sensitization will be viewed and studied in the future.

Ash pollen allergy: reliable detection of sensitization on the basis of IgE to Ole e 1.

SummaryBackground: Alongside hazel, alder and birch pollen allergies, ash pollen allergy is a relevant cause of hay fever during spring in the European region. For some considerable time, ash pollen allergy was not routinely investigated and its clinical relevance may well have been underestimated, particularly since ash and birch tree pollination times are largely the same. Ash pollen extracts are not yet well standardized and diagnosis is therefore sometimes unreliable. Olive pollen, on the other hand, is strongly cross-reactive with ash pollen and is apparently better standardized. Therefore, the main allergen of olive pollen, Ole e 1, has been postulated as a reliable alternative for the detection of ash pollen sensitization.Methods: To determine to what extent specific IgE against Ole e 1 in patients with ash pollen allergy is relevant, we included 183 subjects with ash pollen allergy displaying typical symptoms in March/April and positive skin prick test specific IgE against Ole e 1 (t224) and ash pollen (t25) and various birch allergens (Bet v 1, Bet v 2/v 4) in a retrospective study.Results: A significant correlation was seen between specific IgE against Ole e 1 and ash pollen, but also to a slightly lesser extent between IgE against Ole e 1 and skin prick test with ash pollen, the latter being even higher than IgE and skin prick test both with ash pollen. No relevant correlation was found with birch pollen allergens, demonstrating the very limited cross-reactivity between ash and birch pollen.Conclusion: It appears appropriate to determine specific IgE against Ole e 1 instead of IgE against ash pollen to detect persons with ash pollen allergy. Our findings may also support the idea of using possibly better standardized or more widely available olive pollen extracts instead of ash pollen extract for allergen-specific immunotherapy.

Glutathione-s-transferase is a minor allergen in birch pollen because of restricted release from hydrated pollen grains

Methods bGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses were assessed. 217 sera from birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The allergenicity of bGST was evaluated with IgE-loaded rat basophil leukaemia cells (RBL) expressing the a-chain of the human receptor FceRI. Cross-reactivity of IgE between bGST and GST from house dust mite, Der p 8, was assessed with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen upon hydration were studied by immunoblotting.

The ΙgΕ-epitome viewed through the eyes of human monoclonal ΙgΕ

Background Despite the key role of IgE as the allergen recognizer at the initiation of allergic responses our knowledge on the molecular interactions between human IgE and allergen is limited. To date, most studies aiming at identification of IgE-reactive epitopes have had to rely on polyclonal IgE-preparations, non-human antibodies or antibodies of isotypes other than IgE. However, due to their intrinsic ability to recognize highly relevant, in the context of allergy, epitopes on allergens, allergen-specific human monoclonal IgE, represented by recombinant human antibody fragments, offer an opportunity to decipher the IgE-epitome of major allergens with precision and at high resolution. Such information may guide us in development of novel therapeutic candidates and better diagnostic tools. Methods We have isolated a large pool of antibody variable domain fragments, specific for several major allergens, from the IgE repertoires of allergic donors. Three sets of such binders, targeting the major birch pollen allergen Bet v 1 (n = 4) and the major timothy grass pollen allergens Phl p 1 (n = 5) and Phl p 5 (n = 10), and their interactions with respective allergen have been studied by immunochemical methodologies. Results We have, using our own technology and through collaborations, been able to define multiple non-overlapping IgE-reactive epitopes on all three investigated allergens. Two novel epitopes on Bet v 1 were identified, which allowed for identification of single amino acid residues with crucial importance for the interaction between human IgE and allergens in the PR-10 protein family. Further, it was shown that the relatively small allergen Phl p 5 carries a complex composition of IgE-reactive epitopes, providing an explanation to its high allergenicity. The less complex epitope composition on Phl p 1, on the other hand, with an IgE-binding hot-spot located on the surface of its immunodominant C-terminal domain, allowed us to, in a rational manner, design a hypoallergenic group 1 gras sp ollen allergen fragment, with the potential to be used as a safer alternative to the today used extracts in specific immunotherapy. Conclusion

Random mutagenesis and phage display technology as a tool for identifying ige epitopes of the birch pollen allergen Bet v 1

Background IgE-binding epitopes of the major birch pollen allergen Bet v 1 are dependent on the native structure of the allergen. To date, only a single IgE epitope has been identified. We aimed to identify additional epitopes by directed in vitro evolution of non-IgE-binding structural homologues to proteins carrying single IgE-binding patches. For that purpose, we chose cytokinin-specific binding protein (CSBP) from Vigna radiata (mung bean), a structural homologue of Bet v 1 with only 31% sequence identity, as a template subjected to a combination of random mutagenesis and phage display.

In-print of the environment on the molecular sensitisation profile towards pollen allergens revealed by allergen micro-array

Background The green area in Tashkent-city in Uzbekistan has been re-organized during the last two decades. It covers approximately 35% of the total area and includes classical vegetation present in Central Asia such as saltwort and newly planted species such as Bermuda grass. The knowledge of the molecular sensitization profiles of allergic patients is essential for the correct treatment with allergen specific immunotherapy but has not been established for allergic patients in the Central Asian area. Objective The aim of this study was to determine the IgE-sensitization profile towards pollen allergens in patients with respiratory allergy from Tashkent. Methods Fifty adult patients with allergic rhinitis and/or asthma were tested using an allergen micro-array containing 112 different allergen molecules (ImmunoCAP ISAC; Thermo Scientific). Results We found that the major Bermuda grass pollen allergen, Cyn d 1 and the major saltwort allergen Sal k 1 were most frequently recognized allergens. More than 42% of the patients displayed IgE reactivity to Cyn d 1 and the percentage was even higher in the group suffering from allergic asthma (n=12) (i.e., 66%). Sal k 1 was recognized by 46% of the patients and by 50% of the patients with asthma. The other pollen allergens were recognized less frequently. Interestingly, more than 20% of patients showed IgE cross-reactivity with profilins from grass pollen, weed pollen and birch pollen whereas no patient mounted IgE reactivity to the major birch pollen allergen, Bet v 1. Conclusion The results of the IgE profiling identify grass pollen and in particular Bermuda grass and salkwort as the most important pollen allergen sources in Tashkent. Although approximately 20% of the patients reacted with birch pollen profilin, none of the patients reacted with the major birch pollen allergen, Bet v 1, indicating that these patients had no genuine sensitization to birch. These data show that the molecular sensitization profile towards pollen allergens in Central Asia is an in-print of the local flora and indicates how important allergen micro-array analysis is for the selection of the correct immunotherapy treatment. This study was supported by the Austrian Science Fund (FWF), American Austrian Foundation (AAF) and was performed in the framework of International Network University for Molecular Allergology and Immunology.

Induction of allergen-specific blocking IgG using patch delivered recombinant Bet v 1 in guinea pigs

Background Allergen-specific immunotherapy (SIT) is the only specific, disease-modifying treatment for allergy and may have long-lasting effects. It is given traditionally in the form of multiple subcutaneous injections which makes the treatment inconvenient. Furthermore, systemic allergenadministration may cause severe side effects. Therefor the development of alternative routes for SIT has been a longsought goal. The aim of this study was to investigate if epicutaneous application of recombinant birch pollen allergen Bet v 1 (i.e., patch vaccination) can induce systemic allergen-specific IgG responses with protective activity.

Trimolecular complex between major birch pollen allergen, Bet v 1, monoclonal allergen-specific human IgE and recombinant CD23

CD23 the low affinity receptor for IgE plays an important role in allergic disease because it facilitates allergen presentation to T cells. CD23 is a 45 kDa trans-membrane protein consisting of an alpha-helical stalk and a head domain, which is mainly expressed on B cells. In order to analyze the complex formation between allergen, allergenspecific IgE and CD23 on the molecular level, we expressed four different forms of CD23 in baculovirusinfected SF9 insect cells. Construct A consists of the extracellular part of the molecule, the second construct (B) differs from construct A by a single amino acid exchange in the stalk region to abolish the N-linked glycosylation of CD23. Constructs C and D were smaller molecules consisting mainly of the head domain of the protein. Furthermore we expressed human monoclonal IgE specific for the major birch pollen allergen Bet v 1 in a hybridoma cell line and the Bet v 1 allergen in Escherichia coli. Using circular dichroism analysis we found that all molecules were expressed as folded proteins and gel filtration revealed that the CD23 constructs were monomeric. In ELISA we demonstrated that each of the four CD23 constructs binds monomeric IgE and allergen-IgE complexes in a similar way. Concerning the different CD23 constructs, we observed weaker binding signals when measuring the smaller CD23 molecules. Interestingly, construct B, which was devoid of the N-linked glycosylation site, displayed enhanced IgE binding compared to the glycosylated protein. In conclusion, our results reveal a comparable interaction between isolated IgE as well as IgE-allergen immune complexes with CD23 and provide defined molecules for the detailed structural characterization of the allergenIgE-CD23 interaction. Supported by grants 4605, 4613 and in part by 4604 and P23350-B11 of the Austrian Science Fund (FWF).

Analysis of the IgE epitope profile of soybean allergen Gly m 4

Analysis of the IgE epitope profile of soybean allergen Gly m 4

Development of a novel method for identification and quantification of birch pollen allergens in parallel

Background Determining the content of individual allergens at isoform level has become important for comprehensive standardization and quality control of allergen products as the content of individual allergens has an impact on the potency of allergen extracts. Mass spectrometry (MS) is a dominant and indispensable proteomic tool that has been employed for allergen analysis. Therefore, the main focus of this study was to develop a MS method capable of identifying peptides for quantification of individual Bet v 1 isoforms, and to quantify the entire protein profile including allergenic and non-allergenic proteins of birch pollen in a single run.

Identification of the natural ligand of Bet v 1

The major birch pollen allergen Bet v 1 is the main elicitor of airborne type I allergies and belongs to the family 10 of pathogenesis related proteins. Bet v 1 is the most extensively studied allergen and is well characterized on the biochemical and immunological level. However, its physiological function remained elusive. Here we present the identification of quercetin-3-O-sophoroside (Q3OS) as the natural ligand of Bet v 1. We isolated Q3OS bound to Bet v 1 from mature birch pollen and confirmed its binding by reconstitution of the Bet v 1:Q3OS complex. Fluorescence, UV/VIS as well as HSQC (heteronuclear single quantum coherence) titration experiments and the comparison with model compounds, such as quercetin, proved the specificity of Q3OS binding. The definition of the binding site by NMR together with a computational model give a more detailed understanding and shed light on the physiological function of Bet v 1. We postulate that the binding of Q3OS plays an important but unexplored role during the inflammatory response and the Bet v 1 recognition by IgE.

A molecular model system based on human monoclonal allergen-specific IgE antibodies to study basophil activation

Methods Monoclonal human IgE antibodies specific for the major birch pollen allergen Bet v 1 and for the major grass pollen allergen Phl p 5 and the corresponding allergens as well as a hybrid protein consisting of the Bet v 1 and Phl p 5 allergens were expressed. Using circular dichroism spectroscopy we compared the fold of the hybrid protein with that of the mixture of the two allergens. Rat basophil leukemia cells transfected with the human FceRI receptor were then loaded with the monoclonal IgE antibodies or patients serum containing polyclonal allergen-specific IgE and then exposed to the allergens and the hybrid protein to study basophil activation. Results We found that basophil activation was obtained with a mixture of two monoclonal IgE antibodies and the hybrid protein which increased in magnitude with rising concentrations of allergen-specific IgE antibodies. Thus increases in allergen-specific IgE lead to the release of larger amounts of mediators. When polyclonal IgE from allergic patients was used, basophil activation was obtained already with lower concentrations of the hybrid protein. An augmentation of the number of IgE epitopes thus increased the allergenic activity and thus the “potency” of the allergen. Preliminary titration experiments of the two monoclonal IgE antibodies also suggested that equimolar concentrations of the IgE antibodies with different specificity were most efficient in causing basophil activation, most likely due to the formation of large immune complexes. Conclusion Results obtained in our molecular model for human basophil activation show that the levels of allergen-specific IgE dictate the extent of basophil degranulation and that the number of IgE epitopes determines the potency of an allergen to induce degranulation. The model should be useful to explore the factors determining allergen-induced basophil activation and to study therapeutic strategies controlling this event. This study was supported by grant P23318-B11 and in part by grants P23350-B11 and F4605 of the Austrian Science Fund (FWF).

Sensitisation pattern to birch pollen allergen components in oral allergy syndrome to Rosaceae fruits in patients with spring pollinosis from an East European Sylvosteppe area with low density forests

Methods We evaluated subjects from Southern Romania, a Central European region with temperate continental climate with submediterranean and humid subtropical influences, where sensitization to Betulaceae pollen is less important in pollinosis compared with that to grass or weed pollen. We selected adult patients from the region of sylvosteppe with low density forests dominated by deciduous species, presenting symptoms of rhinoconjunctivitis in March to May and oral allergy syndrome to fresh Rosaceae fruits, allergic reactions in the mouth and throat without systemic symptoms, and having positive skin prick tests to Betulaceae tree pollen and positive prick-to-prick skin tests with fresh intact and unpeeled apple, pear, apricot, peach, cherry or plum. Because allergen components present in Fagales tree pollen and Rosaceae fruits are PR-10 proteins, profilins and isoflavone reductases, we measured the serum levels of specific IgE to European birch pollen and to recombinant allergen components Bet v1 (birch major PR-10 allergen with ribonuclease activity), Bet v2 (birch-pollen profilin), and Bet v6 (birch-pollen isoflavone reductase), using a multiparameter immunoblot test system based on single purified allergen components (SPAC 1).

Bet v 1 and homologous food allergens are similarly processed by antigen-presenting cells but differ in T cell reactivity

Background Various plant foods, e.g. apple and celery, express proteins that are homologues of the major birch-pollen allergen Bet v 1, e.g Mal d 1 and Api g 1. The proteins have 63% and 72% sequence similarity with Bet v 1 and share with it a common 3-dimensional structure. Despite this great molecular similarity, Bet v 1 is the only one among its homologues with the ability to sensitise atopic individuals. The aim of this study was to assess whether differences in the uptake and processing by antigen-presenting cells and in the presentation to T cells could be responsible for Bet v 1’s ability to sensitise.

Nitration of the Birch Pollen Allergen Bet v 1.0101: Efficiency and Site-Selectivity of Liquid and Gaseous Nitrating Agents

Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO–), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O3/NO2). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO–, and Y83 and Y158 for O3/NO2. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O3/NO2 was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein.

Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy

BackgroundCharacterization of IgE-binding epitopes of allergens and determination of their patient-specific relevance is crucial for the diagnosis and treatment of allergy.ObjectiveWe sought to assess the contribution of specific surface areas of the major birch pollen allergen Bet v 1.0101 to binding IgE of individual patients.MethodsFour distinct areas of Bet v 1 representing in total 81% of its surface were grafted onto the scaffold of its homolog, Api g 1.0101, to yield the chimeras Api-Bet-1 to Api-Bet-4. The chimeras were expressed in Escherichia coli and purified. IgE binding of 64 sera from Bet v 1–sensitized subjects with birch pollen allergy was determined by using direct ELISA. Specificity was assessed by means of inhibition ELISA.ResultsrApi g 1.0101, Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4 bound IgE from 44%, 89%, 80%, 78%, and 48% of the patients, respectively. By comparing the amount of IgE binding to the chimeras and to rApi g 1.0101, 81%, 70%, 75%, and 45% of the patients showed significantly enhanced IgE binding to Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4, respectively. The minority (8%) of the sera revealed enhanced IgE binding exclusively to a single chimera, whereas 31% showed increased IgE binding to all 4 chimeras compared with rApi g 1.0101. The chimeras inhibited up to 70% of IgE binding to rBet v 1.0101, confirming the specific IgE recognition of the grafted regions.ConclusionThe Bet v 1–specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is highly patient specific.

Prophylactic and therapeutic vaccination with carrier‐bound Bet v 1 peptides lacking allergen‐specific T cell epitopes reduces Bet v 1‐specific T cell responses via blocking antibodies in a murine model for birch pollen allergy

BackgroundVaccines consisting of allergen-derived peptides lacking IgE reactivity and allergen-specific T cell epitopes bound to allergen-unrelated carrier molecules have been suggested as candidates for allergen-specific immunotherapy.ObjectiveTo study whether prophylactic and therapeutic vaccination with carrier-bound peptides from the major birch pollen allergen Bet v 1 lacking allergen-specific T cell epitopes has influence on Bet v 1-specific T cell responses.MethodsThree Bet v 1-derived peptides, devoid of Bet v 1-specific T cell epitopes, were coupled to KLH and adsorbed to aluminium hydroxide to obtain a Bet v 1-specific allergy vaccine. Groups of BALB/c mice were immunized with the peptide vaccine before or after sensitization to Bet v 1. Bet v 1- and peptide-specific antibody responses were analysed by ELISA. T cell and cytokine responses to Bet v 1, KLH, and the peptides were studied in proliferation assays. The effects of peptide-specific and allergen-specific antibodies on T cell responses and allergic lung inflammation were studied using specific antibodies.ResultsProphylactic and therapeutic vaccination with carrier-bound Bet v 1 peptides induced a Bet v 1-specific IgG antibody response without priming/boosting of Bet v 1-specific T cells. Prophylactic and therapeutic vaccination of mice with the peptide vaccine induced Bet v 1-specific antibodies which suppressed Bet v 1-specific T cell responses and allergic lung inflammation.Conclusion and Clinical RelevanceVaccination with carrier-bound allergen-derived peptides lacking allergen-specific T cell epitopes induces allergen-specific IgG antibodies which suppress allergen-specific T cell responses and allergic lung inflammation.

Secret of the major birch pollen allergen Bet v 1: identification of the physiological ligand

The major birch pollen allergen Bet v 1 is the main elicitor of airborne type I allergies and belongs to the PR-10 family (pathogenesis-related proteins 10). Bet v 1 is the most extensively studied allergen, and is well characterized at a biochemical and immunological level; however, its physiological function remains elusive. In the present study, we identify Q3OS (quercetin-3-O-sophoroside) as the natural ligand of Bet v 1. We isolated Q3OS bound to Bet v 1 from mature birch pollen and confirmed its binding by reconstitution of the Bet v 1-Q3OS complex. Fluorescence and UV-visible spectroscopy experiments, as well as HSQC (heteronuclear single-quantum coherence) titration, and the comparison with model compounds, such as quercetin, indicated the specificity of Q3OS binding. Elucidation of the binding site by NMR combined with a computational model resulted in a more detailed understanding and shed light on the physiological function of Bet v 1. We postulate that the binding of Q3OS to Bet v 1 plays an important, but as yet unclear, role during the inflammation response and Bet v 1 recognition by IgE.

Poster 2001: Generation a hypoallergenic profilin from amaranthus palmeri pollen

Background Amaranthus pollen grains are known to be highly allergenic and a potential cause of respiratory allergic diseases in many countries. Amaranthus palmeri pollen is an important allergenic source in Mexico; however in our country, there is not reported data about the prevalence of these pollens. The first allergenic profilin was identified from birch pollen allergen (Bet v 2) in 1991. Many studies have been described that patients who developed allergies against profilin from pollen commonly presented cross-reactivity against to vegetable food profilin, in particular to different fruits as nuts, melon, orange, and peach, or to vegetables as zucchini, celery, tomatoes and carrots or also latex. The aim of the study is to clone the profilin from A. palmeri pollen and obtain hypoallergenic profilin variant, this variant could be used as a therapeutic.