Subbarayal B.,Medical University of Vienna |
Schiller D.,Paul Ehrlich Institute |
Mobs C.,University of Marburg |
De Jong N.W.,Erasmus Medical Center |
And 8 more authors.
Allergy: European Journal of Allergy and Clinical Immunology | Year: 2013
Background IgE antibodies specific for the major birch pollen allergen frequently cross-react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP-SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross-reactivity of BP-SIT-induced Bet v 1-specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross-reactivity, and IgE-blocking activity of Bet v 1-specific IgG4 antibodies emerging during conventional BP-SIT and whether IgG4-epitopes overlapped with IgE epitopes. Methods IgE and IgG4 levels specific for Bet v 1, Mal d 1, and Cor a 1 were determined in 42 birch pollen-allergic patients before and during BP-SIT. Inhibition of IgE binding was studied by IgE-facilitated antigen-binding assays and basophil activation tests. Furthermore, inhibition of IgE-mediated activation of food allergen-reactive Bet v 1-specific T-cell lines was assessed. Competitive immunoscreening of phage-displayed peptides was applied to select mimotopes recognized by IgE and IgG4 antibodies, respectively. The resulting mimotopes were mapped on the surface of the 3D structure of the allergens using a computer-based algorithm. Results BP-SIT significantly increased Bet v 1- and food allergen-reactive IgG4 antibodies. In parallel, allergen-specific IgE levels decreased significantly. Sera containing food allergen-reactive IgG4 antibodies inhibited IgE binding, basophil activation, and IgE-mediated food allergen-induced T-cell proliferation. Predicted IgE and IgG4 epitopes on all allergens showed high overlap. Conclusion Our results indicate that BP-SIT may induce Bet v 1-specific IgG4 antibodies that cross-react with related food allergens and inhibit IgE binding by epitope competition. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Swoboda I.,Medical University of Vienna |
Twaroch T.E.,Medical University of Vienna |
Vogelsang H.,Clinic for Internal Medicine III |
Kazemi-Shirazi L.,Clinic for Internal Medicine III |
And 11 more authors.
Allergy: European Journal of Allergy and Clinical Immunology | Year: 2011
Background: Cow's milk is one of the most common causes of food allergy. In two-thirds of patients, adverse symptoms following milk ingestion are caused by IgE-mediated allergic reactions, whereas for one-third, the mechanisms are unknown. Aim of this study was to investigate whether patients suffering from non-IgE-mediated cow's milk protein intolerance can be distinguished from persons without cow's milk protein intolerance based on serological measurement of IgG and IgA specific for purified cow's milk antigens. Methods: We determined IgG 1-4 subclass and IgA antibody levels to purified recombinant αS1-casein, αS2-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin in four patient groups by ELISA: Patients with IgE-mediated cow's milk allergy (CMA, n = 25), patients with non-IgE-mediated cow's milk protein intolerance (CMPI, n = 19), patients with gastrointestinal symptoms not associated with cow's milk ingestion (GI, n = 15) and control persons without gastrointestinal problems (C, n = 26). Cow's milk-specific IgE levels were determined by ImmunoCAP. Results: Only CMA patients had IgE antibodies to cow's milk. Cow's milk allergic patients mounted the highest IgG 1 and IgG 4 antibody levels to αS1-casein, αS2-casein, β-casein, κ-casein, and α-lactalbumin. No elevated levels of IgG 4, IgA, and complement-binding IgG subclasses (IgG 1, IgG 2, IgG 3) to purified cow's milk allergens were found within the CMPI patients compared to persons without cow's milk protein intolerance (GI and C groups). Conclusion: Cow's milk protein intolerant patients cannot be distinguished from persons without cow's milk protein intolerance on the basis of IgG subclass or IgA reactivity to cow's milk allergens. © 2011 John Wiley & Sons A/S.
Geroldinger-Simic M.,Medical University of Vienna |
Zelniker T.,Medical University of Vienna |
Aberer W.,Medical University of Graz |
Ebner C.,Allergy Clinic Reumannplatz |
And 8 more authors.
Journal of Allergy and Clinical Immunology | Year: 2011
Background: Patients with birch pollen allergy often develop allergic reactions to plant foods. Objective: To evaluate the prevalence, main symptoms, and triggers of birch pollen-related food allergy and the role of food-specific IgG4 antibodies in food tolerance. Methods: Food-induced symptoms were evaluated in 225 individuals with birch pollen allergy by using a standardized questionnaire. IgE and IgG4 levels specific for the major birch pollen allergen Bet v 1 and birch profilin Bet v 2 and the Bet v 1 homologs in apple (Mal d 1) and hazelnut (Cor a 1) were quantified by ImmunoCAP. Mock-treated and IgG-depleted sera from patients tolerating hazelnuts in food challenges were compared for their inhibitory activity for binding of Cor a 1-IgE complexes to B cells. Results: In total, 73% of the study population experienced food allergy, which was perennial in 86% of the affected individuals. The oral allergy syndrome was the main clinical manifestation. However, more than 58% of the patients also experienced food-induced rhinoconjunctivitis. Apples and hazelnuts were identified as the most frequent triggers. Food allergy correlated with IgE reactivity to Bet v 1 but not to Bet v 2. Mal d 1-specific and Cor a 1-specific IgG4/IgE ratios were significantly higher in food-tolerant individuals than individuals with food allergy. Sera from IgG4-positive food-tolerant patients possessed IgG-dependent IgE-inhibitory activity. Conclusion: Birch pollen-related food allergy is highly prevalent and often perennial. High food allergen-specific IgG4/IgE ratios seem associated with food tolerance, potentially because specific IgG4 blocks IgE binding to food allergens. Thus, the presence of food allergen-specific IgG4 antibodies is no diagnostic marker for birch pollen-related food allergy. © 2010 American Academy of Allergy, Asthma and Immunology.
PubMed | Federal University of Rio de Janeiro, Karl Landsteiner University for Medical science, University of Innsbruck, Allergy Clinic Reumannplatz and Medical University of Vienna
Type: Journal Article | Journal: PloS one | Year: 2015
Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients sera to all three -parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.
Geroldinger-Simic M.,Medical University of Vienna |
Kinaciyan T.,Medical University of Vienna |
Nagl B.,Medical University of Vienna |
Baumgartner-Durchschlag U.,Biomay AG |
And 8 more authors.
Journal of Allergy and Clinical Immunology | Year: 2013
Background: Antibodies and T cells specific for the major birch pollen allergen Bet v 1 cross-react with structurally related food allergens, such as Mal d 1 in apple. Objective: We sought to evaluate the effects of oral uptake of Mal d 1 on the allergen-specific immune response in patients with birch pollen allergy. Methods: Patients received 50 μg of rBet v 1 sublingually on 2 consecutive days outside of the birch pollen season. One year later, equal amounts of rMal d 1 were administered. Blood samples were collected before and after oral exposure, as well as before and after the intermediate birch pollen season. Allergen-specific IgE levels were determined by using ImmunoCAP. Proliferation of allergen-stimulated PBMCs was assessed, as well as the expression of IL-5, IL-13, IL-10, IFN-γ, and forkhead box protein 3 (Foxp3) in isolated T cells (real-time PCR). Allergen-specific T-cell lines were analyzed for epitope recognition. Results: Orally administered Bet v 1 transiently reduced Bet v 1-specific serum IgE levels, as well as Bet v 1- and Mal d 1-induced T-cell proliferation, and enhanced the expression of IL-5, IL-10, and Foxp3. Orally applied Mal d 1 significantly decreased Bet v 1- and Mal d 1-specific IgE levels and induced IL-5 and IL-10 but no Foxp3 expression. In contrast to Bet v 1, Mal d 1 triggered IFN-γ production and T cells with a different epitope repertoire. Inhalation of birch pollen significantly enhanced allergen-specific IgE levels, T-cell proliferation, and IL-5, IL-10, IL-13, and Foxp3 expression. Conclusion: Two sublingual administrations of 50 μg of Mal d 1 were well tolerated and induced transient immune responses seen during peripheral tolerance development. Thus recombinant Mal d 1 might be suitable and relevant for sublingual treatment of birch pollen-related apple allergy. © 2012 American Academy of Allergy, Asthma & Immunology.
Vejvar E.,Christian Doppler Laboratory |
Himly M.,Christian Doppler Laboratory |
Briza P.,Christian Doppler Laboratory |
Eichhorn S.,Christian Doppler Laboratory |
And 4 more authors.
Molecular Nutrition and Food Research | Year: 2013
Scope: Apium graveolens represents a relevant food allergen source linked with severe systemic reactions. We sought to identify an IgE-binding nonspecific lipid transfer protein (nsLTP) in celery tuber. Methods and results: A low molecular weight protein exclusively present in celery tuber was purified and designated Api g 6. The entire protein sequence was obtained by MS and classified as member of the nsLTP2 family. Api g 6 is monomeric in solution with a molecular mass of 6936 Da. The alpha-helical disulfide bond-stabilized structure confers tremendous thermal stability (Tm > 90°C) and high resistance to gastrointestinal digestion. Endolysosomal degradation demonstrated low susceptibility and the presence of a dominant peptide cluster at the C-terminus. Thirty-eight percent of A. graveolens allergic patients demonstrated IgE reactivity to purified natural Api g 6 in ELISA and heat treatment did only partially reduce its allergenic activity. No correlation in IgE binding and limited cross-reactivity was observed with Api g 2 and Art v 3, nsLTP1 from celery stalks and mugwort pollen. Conclusion: Api g 6, a novel nsLTP2 from celery tuber represents the first well-characterized allergen in this protein family. Despite similar structural and physicochemical features as nsLTP1, immunological properties of Api g 6 are distinct which warrants its inclusion in molecule-based diagnosis of A. graveolens allergy. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Ackerbauer D.,Medical University of Vienna |
Bublin M.,Medical University of Vienna |
Radauer C.,Medical University of Vienna |
Varga E.-M.,Medical University of Graz |
And 7 more authors.
International Archives of Allergy and Immunology | Year: 2015
Background: Peanut allergy develops after primary sensitization to peanut allergens and/or IgE cross-sensitization with homologous allergens from various plants. Therefore, heterogeneous patterns of sensitization to individual peanut allergens are observed in different countries. The aim of this study was to examine the IgE sensitization patterns of Austrian peanut-allergic patients. Methods: Sera from 65 peanut-allergic patients and 20 peanut-tolerant atopics were obtained in four Austrian allergy clinics. Sensitization patterns against peanut allergens Ara h 1-3, 6, 8 and 9 were identified by ImmunoCAP and ImmunoCAP ISAC. Results: Austrian peanut-allergic patients were sensitized to Ara h 2 and 6 (71%), followed by Ara h 1 (62%), Ara h 8 (45%), Ara h 3 (35%) and Ara h 9 (11%). All sera containing Ara h 2-specific IgE were also positive for Ara h 6, with Ara h 6-specific IgE levels significantly (p < 0.05) higher compared with Ara h 2. Twelve percent displayed IgE reactivity exclusively to Ara h 8. Peanut extract and Ara h 8 showed low diagnostic specificities of 25 and 10%, respectively. The other peanut allergens showed 100% specificity. Diagnostic sensitivities determined by ImmunoCAP ISAC and ImmunoCAP were highly similar for Ara h 2, 3 and 8. Conclusions: The majority of symptomatic peanut-allergic patients are sensitized to Ara h 2 and Ara h 6. In peanut-symptomatic patients with additional birch pollen allergy, other peanut allergens, especially Ara h 8, should be tested when IgE reactivity to Ara h 2 is absent. © 2015 S. Karger AG, Basel.
Hochwallner H.,Medical University of Vienna |
Schulmeister U.,Medical University of Vienna |
Swoboda I.,Christian Doppler Laboratory |
Balic N.,Medical University of Vienna |
And 14 more authors.
Clinical and Experimental Allergy | Year: 2010
Background Cow's milk is one of the most common causes of food allergy affecting approximately 2.5% of infants in the first years of their life. However, only limited information regarding the allergenic activity of individual cow's milk allergens is available.Objective To analyse the frequency of IgE reactivity and to determine the allergenic activity of individual cow's milk allergens.Methods A nitrocellulose-based microarray, based on purified natural and recombinant cow's milk allergens was used to determine IgE reactivity profiles using sera from 78 cow's milk-sensitized individuals of varying ages. The allergenic activity of the individual allergens was tested using patients' sera for loading rat basophil leukaemia cells (RBL) expressing the α-chain of the human receptor FcεRI.Results Using the microarray and the RBL assay, cow's milk allergens were assessed for frequency of IgE recognition and allergenic activity. Moreover, the RBL assay allowed distinguishing individuals without or with mild clinical reactions from those with severe systemic or gastrointestinal symptoms as well as persons who grew out cow's milk allergy from those who did not.Conclusions Component-resolved testing using milk allergen microarrays and RBL assays seems to provide useful additional diagnostic information and may represent a basis for future forms of prophylactic and therapeutic strategies for cow's milk allergy. © 2010 Blackwell Publishing Ltd.
PubMed | Allergy Clinic Floridsdorf, Allergy Clinic Reumannplatz, Allergy Clinic Rennweg and Medical University of Vienna
Type: Journal Article | Journal: Allergy | Year: 2016
IgG to galactose--1,3-galactose (-gal) are highly abundant natural antibodies (Ab) in humans. -Gal-specific IgE Ab cause a special form of meat allergy characterized by severe systemic reactions 3-7 h after consumption of red meat. We investigated 20 patients who experienced such reactions and characterized their -gal-specific IgE and IgG responses in more detail.-Gal-specific IgE was determined by ImmunoCAP. IgE reactivity to meat extract and bovine gamma globulin (BGG) was assessed by immunoblotting and ELISA, respectively. In some experiments, sera were pre-incubated with -gal or protein G to deplete IgG Ab. -Gal-specific IgGIn immunoblots, BGG was the most frequently recognized meat protein. Binding of IgE and IgG to BGG was confirmed by ELISA and completely abolished after pre-incubation with -gal. Neither the depletion of autologous -gal-specific IgG Ab nor the addition of -gal-specific IgG Ab from nonallergic individuals changed the IgE recognition of BGG of meat-allergic patients. Meat-allergic patients showed significantly higher -gal-specific IgG1 and IgG3 Ab than nonallergic individuals, whereas the latter showed significantly higher levels of -gal-specific IgG4 Ab.Patients with delayed meat allergy display IgE and IgG Ab that selectively recognize the -gal epitope on BGG. Their enhanced -gal-specific IgE levels are accompanied by high levels of -gal-specific IgG1 devoid of IgE-blocking activity. This subclass distribution is atypical for food allergies and distinct from natural -gal IgG responses in nonallergic individuals.
PubMed | Paracelsus Medical University, Allergy Clinic Reumannplatz, Christian Doppler Laboratory and University of Salzburg
Type: | Journal: The Journal of allergy and clinical immunology | Year: 2016
Allergy vaccines should be easily applicable, safe and efficacious. For Bet v 1-mediated birch pollen and associated food allergies a single wildtype allergen does not provide a complete solution.Thus, we aimed to combine immunologically relevant epitopes of Bet v 1 and the two clinically most important related food allergens from apple and hazelnut to a single hybrid protein, termed MBC4.After identification of T cell epitope-containing parts on each of the three parental allergens, the hybrid molecule was designed to cover relevant epitopes and evaluated in silico. Thereby, a mutation was introduced into the hybrid sequence, which should alter the secondary structure without compromising immunogenic properties of the molecule.MBC4 as well as the parental allergens were purified to homogeneity. Analyses of secondary structure elements revealed substantial changes rendering the hybrid de facto non-reactive with patients serum IgE. Nevertheless, the protein was monomeric in solution. MBC4 was able to activate T cell lines from birch pollen allergic donors and from mice immunized with the parental allergens. Moreover, upon immunization of mice and rabbits MBC4 induced cross-reactive IgG antibodies, which were able to block the binding of human serum IgE.Directed epitope rearrangements combined with a knowledge-based structural modification resulted in a protein unable to bind IgE from allergic patients. Still, properties to activate specific T cells or to induce blocking antibodies were conserved. This suggests that MBC4 is a suitable vaccine candidate for the simultaneous treatment of Bet v 1- and associated food allergies.