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ABSTRACT: The Toll-like receptor (TLR) 5 agonist flagellin is associated with immunomodulatory functions.
We sought to investigate whether Listeria monocytogenes-derived flagellin A (flaA) can modulate ovalbumin (OVA)-specific T-cell responses and prevent OVA-induced intestinal allergy.
Bone marrow-derived myeloid dendritic cells from BALB/c, C57BL/6, or TLR signaling-deficient (MyD88(-/-)) mice were stimulated with rOVA, rflaA, rflaA plus rOVA, or a recombinant fusion protein consisting of rflaA and rOVA (rflaA:OVA). The immunomodulating properties of rflaA plus rOVA and rflaA:OVA were investigated by means of DC-T-cell coculture with CD4(+) T cells from OVA-T-cell receptor transgenic or OVA/alum-immunized mice. rflaA:OVA was applied as a prophylactic and therapeutic vaccine in a murine model of intestinal allergy.
rflaA:OVA induced upregulation of TLR5 and dose-dependent IL-6 and IL-10 secretion by myeloid dendritic cells. IL-10 contributed to repressing IL-4 and IFN-γ secretion by OVA-T-cell receptor transgenic CD4(+) T cells. Moreover, rflaA:OVA suppressed CD4(+) T cells derived from T(H)2-biased mice on OVA/alum immunization. In a murine model of intestinal allergy, prophylactic vaccination with rflaA:OVA reduced T-cell activation. Protection from intestinal allergy included suppression of OVA-specific IgE while inducing OVA-specific IgG(2a). Equimolar amounts of rflaA or rOVA provided alone or as a mixture did not have comparable effects. Moreover, therapeutic vaccination was shown to reduce allergic symptoms and T-cell activation in the spleen.
The rflaA:OVA fusion protein showed strong TLR-mediated immunomodulating capacities probably attributed by the proximity of adjuvant and allergen, leading to the prevention of intestinal allergy in a murine disease model. Therefore recombinant flaA:allergen fusion proteins are promising vaccine candidates for intervention in patients with IgE-mediated allergy.
The Journal of allergy and clinical immunology 08/2011; 128(6):1340-1348.e12. · 9.17 Impact Factor
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Kerstin Bauermeister, Andrea Wangorsch,
Lorenza Perono Garoffo,
Andreas Reuter,
Amedeo Conti,
Steve L Taylor,
Jonas Lidholm,
Asa Marknell Dewitt,
Ernesto Enrique,
Stefan Vieths,
Thomas Holzhauser,
Barbara Ballmer-Weber,
Gerald Reese
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ABSTRACT: Published data on crustacean allergens are incomplete. The identification of tropomyosin (TM), arginine kinase (AK), sarcoplasmic Ca-binding protein (SCP) and myosin light chain (MLC) as shrimp allergens are all important contributions but additional allergens are required for the development of a complete set of reagents for component resolved diagnosis and the exploration of novel vaccination strategies.
The North Sea shrimp (Crangon crangon), which is frequently consumed in Europe, served as a model organism in this study. TM and AK were directly cloned from mRNA based on sequence homology and produced as recombinant proteins. Additional IgE-reactive proteins were isolated by preparative SDS-PAGE and identified by mass spectrometry and corresponding cDNAs were cloned and expressed in E. coli. The relevance of the 6 cloned crustacean allergens was confirmed with sera of 31 shrimp-allergic subjects, 12 of which had a positive double-blind, placebo-controlled food challenge (DBPCFC) to shrimp and 19 a convincing history of food allergy to shrimp, including 5 cases of anaphylaxis. Quantitative IgE measurements were performed by ImmunoCAP.
Six recombinant crustacean proteins: TM, AK, SCP, a novel MLC, troponin C (TnC), and triosephosphate isomerase (TIM) bound IgE in ImmunoCAP analysis. Specific IgE to at least one of these single shrimp allergens was detected in 90% of the study population, thus the in vitro diagnostic sensitivity was comparable to that of shrimp extract (97%). In 75% of the subjects, the combined technical sensitivity was similar to or greater with single shrimp allergens than with natural shrimp extract.
We identified six IgE-binding proteins from C. crangon, three of which have not before been described as allergens in crustaceans. This extensive panel of shrimp allergens forms a valuable asset for future efforts towards the identification of clinically relevant biomarkers and as a basis to approach patient-tailored immunotherapeutic strategies.
Molecular Immunology 07/2011; 48(15-16):1983-92. · 2.90 Impact Factor
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Ana I Sancho, Andrea Wangorsch,
Bettina M Jensen,
Andrew Watson,
Yuri Alexeev,
Phil E Johnson,
Alan R Mackie,
Angela Neubauer,
Gerald Reese,
Barbara Ballmer-Weber,
Karin Hoffmann-Sommergruber,
Per S Skov,
Stefan Vieths,
Elisabeth N Clare Mills
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ABSTRACT: Four Bet v 1 homologous food allergens from celeriac (rApi g 1), apple (rMal d 1), peach (rPru p 1) and hazelnut (rCor a 1), were used to probe the structural responsiveness of the Bet v 1 scaffold to gastric digestion conditions and its impact on allergenicity.
Low pH induced conformational changes of all homologues, which was reduced at physiological ionic strength for all except rPru p 1 as observed by circular dichroism (CD)-spectroscopy. The homologues were rapidly digested by pepsin, losing their IgE binding activity, although the kinetics and patterns of digestion varied subtly between homologues, rApi g 1 being the most stable. We have demonstrated for the first time that gastric phosphatidyl-choline (PC) induced conformational changes in all homologues but only rMal d 1 penetrated the PC vesicles as detected by fluorescence polarization, slowing its digestion and retaining more of its allergenic activity. PC enhanced basophil activation of all digested allergens except rApi g 1.
The Bet v 1 scaffold is generally susceptible to low pH and pepsinolysis and interacts with PC vesicles, properties which can explain effects of the gastric environment on their allergenicity. These data show the importance of including surfactants in model digestion systems.
Molecular Nutrition & Food Research 07/2011; 55(11):1690-9. · 4.30 Impact Factor
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Melanie Albrecht,
Yvonne Kühne,
Barbara K Ballmer-Weber,
Wolf-Meinhard Becker,
Thomas Holzhauser,
Iris Lauer,
Andreas Reuter,
Stefanie Randow,
Sabine Falk, Andrea Wangorsch,
Jonas Lidholm,
Gerald Reese,
Stefan Vieths
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ABSTRACT: Analysis of IgE antibody binding to epitopes provides information for food allergy diagnosis and management and construction of hypoallergenic candidate vaccines, but the contribution of sequential epitopes to functionally relevant IgE binding is not fully understood.
We sought to study the impact of IgE-binding peptides described as major sequence epitopes in the literature on IgE-binding capacity of 2 selected food allergens.
IgE-binding peptides of the food allergens Ara h 2 (peanut) and Pen a 1 (shrimp) were identified. Synthetic soluble peptides representing the identified sequences were assessed for their capacity to inhibit IgE binding to the parent allergens by means of ELISA and in mediator release assay. The IgE-binding capacity of unfolded recombinant (r) Ara h 2 was analyzed. A hybrid tropomyosin carrying the IgE-binding regions of Pen a 1 grafted into the structural context of the nonallergenic mouse tropomyosin was applied in ELISA inhibition experiments and ImmunoCAP analysis.
Although IgE-binding peptides representing sections of the allergen sequences were detected, no relevant capacity to inhibit the IgE binding to the parent allergen in ELISA or basophil activation test was observed. Unfolded rAra h 2 showed reduced IgE-binding capacity compared with folded rAra h 2 and failed to elicit mediator release. Hybrid tropomyosin bound less IgE than rPen a 1 in ImmunoCAP analysis and revealed marginal inhibitory capacity.
Peptides identified as major sequence epitopes on Pen a 1 and Ara h 2 show little contribution to the IgE binding of the allergens studied.
The Journal of allergy and clinical immunology 09/2009; 124(2):328-36, 336.e1-6. · 9.17 Impact Factor
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ABSTRACT: Carrot allergy is caused by primary sensitization to birch pollen. Continuous carrot exposure results in additional Dau c 1-specific allergic responses. Thus, immunotherapy with birch pollen may not improve the food allergy.
Evaluation of mutation and oligomerization of the major carrot allergen, Dau c 1, in regard to alteration of antibody binding capacities, structure, and the ability to induce blocking IgG antibodies.
Measurement of IgE reactivities to monomers, dimers of wild-type and mutant Dau c 1.0104 and Dau c 1.0201, and Dau c 1.0104 trimer, their ability to induce blocking antibodies in mice, and their allergenic potency by histamine release.
The reactivity of human IgE to the mutant dimer was reduced on average by 81%. Sera of immunized Balb/c mice showed specific IgG similar to the human IgE antibody response; Dau c 1.01 was more antigenic than Dau c 1.02. Both wild-type and mutant Dau c 1 variants induced cross-reacting IgG, which blocked binding of human IgE. The mutants were more antigenic than the wild-type forms, and the dimers induced higher IgG responses in mice than the monomers. The results of the histamine release experiments corroborated the findings of the antibody binding studies.
Destruction of native conformation rather than oligomerization is the appropriate strategy to reduce the allergenicity of Bet v 1-homologous food allergens.
The dimer composed of mutants of Dau c 1.0104 and Dau c 1.0201 is a promising candidate vaccine for treatment of carrot allergy because of its high immunogenicity and drastically reduced allergenicity.
Journal of Allergy and Clinical Immunology 05/2007; 119(4):944-51. · 11.00 Impact Factor
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ABSTRACT: For better understanding the cross-reactivity between the major birch pollen and celery allergens, Bet v 1 and Api g 1, respectively, putative epitope areas and structurally important positions for IgE-binding of the isoforms Api g 1.01 and Api g 1.02 were point mutated. The IgE binding capacities were measured in ELISA, the IgE cross-reactivity between the isoforms, mutants and Bet v 1 investigated by ELISA-inhibition experiments with serum pools from patients with confirmed celery allergy (DBPCFC). Api g 1.01 displayed a clearly higher frequency and capacity of IgE binding than Api g 1.02. In Api g 1.01, substitution of lysine against glutamic acid at amino acid position 44, a key residue of the Bet v 1 "P-loop", increased the IgE-binding properties. Structural instability due to proline insertion at position 111/112 resulted in loss of IgE binding of Api g 1.01, but not of Api g 1.02. Between Api g 1.01 and Api g 1.02 only partial cross-reactivity was seen. The data suggest that the IgE epitopes of the two isoforms are distinct and that in contrast to Api g 1.01, the "P-loop" region plays an important role for IgE binding of celery allergic subjects to Api g 1.02. Understanding and investigation of the molecular mechanisms in celery allergy is an important step to generate hypoallergenic proteins for safe and efficacious immunotherapy of food allergy.
Molecular Immunology 05/2007; 44(10):2518-27. · 2.90 Impact Factor
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ABSTRACT: Birch (Betula verrucosa) pollen-associated food allergy is a well-characterized syndrome, which is due to the cross-reactivity of IgE antibodies to homologous allergens in various foods. One crossreacting area on the major birch pollen allergen Bet v 1 and its homologue in cherry (Prunus avium) Pru av 1 has already been identified. This is the so-called 'P-loop' region, which encompasses amino acid residues around position 45 and is found on the two virtually identical tertiary protein structures. We tried to determine an additional IgE cross-reacting patch on Pru av 1 and Bet v 1. The putative IgE-binding region on Pru av 1 was localized with a mAb (monoclonal antibody) that was generated against Bet v 1, and cross-reacts with several Bet v 1 homologues in food and inhibits the binding of patients' IgE to Pru av 1. mAb reactivity pattern was analysed and amino acid positions 28 and 108 of Pru av 1 were selected and mutated by site-directed mutagenesis. The Pru av 1 mutants were produced as recombinant proteins and characterized for their folding, mAb- and IgE-binding capacity and allergenic potency with a cellular assay using the humanized rat basophilic leukaemia cell line RBL-25/30. Amino acid position 28 is involved in a second major IgE-binding region on Pru av 1 and probably on Bet v 1. The identification of this second major IgE-binding region is an essential prerequisite to understand the phenomenon of cross-reactivity and its clinical consequences, and to produce hypoallergenic proteins for an improved immunotherapy of type I allergy.
Biochemical Journal 02/2005; 385(Pt 1):319-27. · 4.90 Impact Factor
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ABSTRACT: The influence of thermal processing and nonenymatic as well as polyphenoloxidase-catalyzed browning reaction on the allergenicity of the major cherry allergen Pru av 1 was investigated. After thermal treatment of the recombinant protein rPru av 1 in the absence or presence of carbohydrates, SDS-PAGE, enzyme allergosorbent tests, and inhibition assays revealed that thermal treatment of rPru av 1 alone did not show any influence on the IgE-binding activity of the protein at least for 30 min, thus correlating well with the refolding of the allergen in buffer solution as demonstrated by CD spectroscopic experiments. Incubation of the protein with starch and maltose also showed no effect on IgE-binding activity, whereas reaction with glucose and ribose and, even more pronounced, with the carbohydrate breakdown products glyceraldehyde and glyoxal induced a strong decrease of the IgE-binding capacity of rPru av 1. In the second part of the study, the effect of polyphenoloxidase-catalyzed oxidation of polyphenols on food allergen activity was investigated. Incubation of rPru av 1 with epicatechin in the presence of tyrosinase led to a drastic decrease in IgE-binding activity of the protein. Variations of the phenolic compound revealed caffeic acid and epicatechin as the most active inhibitors of the IgE-binding activity of rPru av 1, followed by catechin and gallic acid, and, finally, by quercetin and rutin, showing significantly lower activity. On the basis of these data, reactive intermediates formed during thermal carbohydrate degradation as well as during enzymatic polyphenol oxidation are suggested as the active chemical species responsible for modifying nucleophilic amino acid side chains of proteins, thus inducing an irreversible change in the tertiary structure of the protein and resulting in a loss of conformational epitopes of the allergen.
Journal of Agricultural and Food Chemistry 07/2004; 52(12):4002-7. · 2.82 Impact Factor
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ABSTRACT: Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu45 to Trp45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp45, demonstrating that the side chain of Glu45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala112 nor deletion of the C-terminal residues 155-159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the cross-reactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy.
Biochemical Journal 12/2003; 376(Pt 1):97-107. · 4.90 Impact Factor
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Allergy 10/2003; 56(s67):78 - 82. · 6.27 Impact Factor
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Arbeiten aus dem Paul-Ehrlich-Institut (Bundesamt für Sera und Impfstoffe) zu Frankfurt a.M 02/2003;
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ABSTRACT: Profilin is a panallergen which is recognised by IgE from about 20% of birch pollen- and plant food-allergic patients. Little is known about epitope diversity among these homologous proteins, and about the correlation between IgE-cross-reactivity and allergenic reactivity. Plant food profilins from pear (Pyr c 4) and cherry (Pru av 4) were cloned by polymerase chain reaction and produced in Escherichia coli BL21. The profilins were purified as non-fusion proteins by affinity chromatography on poly-(l-proline)-Sepharose and characterized by immunoblotting, IgE-inhibition experiments and histamine release assays. The coding regions of the cDNA of pear and cherry profilin were identified as a 393 bp open reading frame. The deduced amino acid sequences showed high identities with birch pollen profilin Bet v 2 (76–83%) and other allergenic plant profilins. Pyr c 4 and Pru av 4 were investigated for their immunological properties in comparison with profilins from celery (Api g 4) and birch pollen (Bet v 2). Fourty-three of 49 patients (88%), preselected for an IgE-reactivity with Bet v 2 showed specific IgE-antibodies to the recombinant pear protein, 92% of the sera were positive with the recombinant cherry allergen and 80% of the sera were reactive with the celery protein. Inhibition experiments showed a strong cross-reactivity of IgE with profilins from plant food and birch pollen. However, IgE binding profiles also indicated the presence of epitope differences among related profilins. All investigated profilins, Pyr c 4, Pru av 4, Api g 4 and Bet v 2, presented almost identical allergenic properties in cellular mediator release tests. Therefore, cross-reactivities between related profilins may explain pollen-related allergy to food in a minority of patients. The nucleotide sequences reported have been submitted to the Genbank database under accession numbers AF129424 (Pyr c 4) and AF129425 (Pru av 4).
Journal of chromatography. B, Biomedical sciences and applications 06/2001;
