[Recombinant allergens; allergy vaccine in the future?. Interview by Dr. Barbara Scholtissek].
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Allergens↗
Biomedical subjects
R Valenta
Publications and source records attributed to R Valenta.
Skin testing with wild-type recombinant birch pollen allergens and hypoallergenic modified molecules.
UNLABELLED: Our aim was to compare the allergenic activity of wild type rBet v 1 with recombinant Bet v 1 derivatives (rBet v 1 fragments, dimer and trimer) with potentially reduced anaphylactic activity by skin testing in a French population. METHODS: Among the 36 birch pollen allergic patients included in the study, 29 were tested by skin prick testing and 30 by intradermal injections with purified monosubstances: rBet v 1 fragments (F1: aa1-74 and F2: aa75-160), Bet v 1 dimer and trimer. Intradermal tests were performed by the endpoint intradermal titration method. Tests were performed over a period of 6 months (before, during and after birch pollen season). RESULTS: All patients showed lower reactivity with the modified rBet v 1 allergens, both in skin prick- and intradermal tests. In 25 and 23 out of 29 patients the lowest concentration of fragment 1 and 2 respectively, resulting in a positive prick test was 100 fold higher than the lowest concentration of monomer resulting in a positive prick test. For dimer it was 100 fold or more in 25 out of 29 patients, and for trimer it was 100 fold or superior in 26 out of 29 patients. By intradermal testing, the end point concentration was 160 fold higher for trimer than for monomer in 24 patients and 40 fold higher in 5 patients. For the 2 fragments the end point concentration was 160 fold higher in 20 out of 22 patients. CONCLUSION: rBet v 1 fragments and trimer may represent candidate molecules for immunotherapy of birch pollen allergy with reduced risk of anaphylactic side effects.
Allergens↗
Large scale production and quality criteria of recombinant allergens.
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Allergens↗
Molecular and immunologic characterization of a highly cross-reactive two EF-hand calcium-binding alder pollen allergen, Aln g 4: structural basis for calcium-modulated IgE recognition.
Serum IgE was used to isolate a cDNA coding for a 9.4-kDa two EF-hand calcium-binding allergen, Aln g 4, from a lambda gt11 expression cDNA library constructed from alder (Alnus glutinosa) pollen. rAln g 4 was overexpressed in Escherichia coli and purified to homogeneity. It reacted with serum IgE from 18% of pollen-allergic patients (n = 122); shared IgE epitopes with homologous allergens present in tree, grass, and weed pollens; and thus belongs to a family of highly cross-reactive pollen allergens. Exposure of two E. coli-expressed rAln g 4 fragments comprising amino acids 1-41 and 42-85 to patients' IgE Abs, as well as to a rabbit antiserum raised against purified rAln g 4, indicated that most of the B cell epitopes reside in the N-terminal portion of the protein. IgE recognition of Aln g 4 was strongly modulated by the presence or absence of calcium. Circular dichroism analysis of rAln g 4 revealed that the protein consisted mostly of alpha helical secondary structure and possessed a remarkable thermal stability and refolding capacity, a property that was greatly reduced after calcium depletion. Circular dichroism analysis of the calcium-bound and apo form of rAln g 4 indicated that calcium-induced modulation of IgE binding could be due to changes in the protein conformation. Purified rAln g 4 elicited dose-dependent basophil histamine release and immediate type skin reactions in sensitized patients. It may hence be useful for allergy diagnosis and for specific immunotherapy.
Allergens↗
Immunization with purified natural and recombinant allergens induces mouse IgG1 antibodies that recognize similar epitopes as human IgE and inhibit the human IgE-allergen interaction and allergen-induced basophil degranulation.
Molecular characterization of allergens by recombinant DNA technology has made rapid progress in the recent few years. In the present study we immunized mice with aluminum hydroxide-adsorbed purified recombinant major timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5), dog albumin, a major animal dander allergen, and proteins with low (beta-lactoglobulin) or no (ribulose diphosphate carboxylase) allergenic potential in humans. Allergens that bind high levels of IgE in humans (Phl p 1, Phl p 5, dog albumin) induced high IgE and IgG1 levels in mice, whereas proteins with little or no allergenic activity in humans failed to induce significant IgE and IgG1 levels in mice. Continuous immunization for a period of 27 wk resulted in the production of mouse IgG1 Abs that recognized recombinant allergen fragments/epitopes defined by IgE Abs of allergic patients. As a consequence, allergen-specific mouse Abs strongly inhibited human IgE binding to the allergens and suppressed the allergen-induced histamine release from human basophils. In summary, our data indicate that 1) the allergenic potency of a protein may be related to its overall immunogenicity and 2) prolonged immunization with single purified recombinant allergens induces protective IgG Abs. The presented experimental in vivo/in vitro system allows the evaluation of Ag preparations (e.g., recombinant allergens) to be used for immunotherapy in humans.
Allergens↗
Human monoclonal IgG antibodies derived from a patient allergic to birch pollen as tools to study the in situ localization of the major birch pollen allergen, Bet v 1, by immunogold electron microscopy.
BACKGROUND: Bet v 1, the major birch pollen allergen, and related allergens present in various tree pollens, fruits, and vegetables represent a family of important cross-reactive allergens. Although the DNA, deduced amino-acid sequence, and structure of Bet v 1 have been determined, little is known regarding its biologic functions. OBJECTIVE: Human monoclonal antibodies derived from an individual allergic to birch pollen were used for refined ultrastructural localization of Bet v 1 in birch pollen grains to gain information regarding the allergen distribution and its possible biologic function. These data were to be supplemented by sequence analyses. METHODS: Ultrathin sections of anhydrously prepared birch pollen grains were incubated with human monoclonal antibodies (BAB1, BAB2, and BAB4). The binding sites were visualized in the transmission electron microscope by gold-conjugated anti-human IgG antibodies. RESULTS: In the cytoplasm of the birch pollen grain, human monoclonal antibodies bound to ribosome-rich areas and to pollen nuclei. Sequence analysis of Bet v 1 and homologous allergens identified a highly conserved p-loop motif in these proteins, which is typically found in nucleotide-binding proteins. CONCLUSIONS: Immunolocalization of Bet v 1 to ribosome-rich areas and the nucleus would be consistent with a highly conserved biologic function of Bet v 1 and homologous proteins as nucleotide binding proteins and explains why this group of plant proteins represents highly cross-reactive plant allergens against which many type I patients are sensitized.
Allergens↗
Parvalbumin, a cross-reactive fish allergen, contains IgE-binding epitopes sensitive to periodate treatment and Ca2+ depletion.
BACKGROUND: Type I allergy to fish is a severe health problem in countries in which a large percentage of the population derive income from fishing. OBJECTIVE: The aim of the study was to characterize cross-reactive IgE-binding components in six different fish species (cod, tuna, salmon, perch, carp, and eel). The effect of reducing extraction conditions, periodate treatment, and depletion of Ca2+ on binding of IgE to the allergens was investigated. METHODS: Extracts were prepared under nonreducing and reducing conditions. IgE-binding components were characterized by IgE immunoblotting, and cross-reactive epitopes were studied by IgE-immunoblot inhibition experiments. To reveal calcium-sensitive or carbohydrate-containing epitopes, nitrocellulose-blotted extracts were exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and periodate. RESULTS: Sera from all patients allergic to fish (n = 30) displayed IgE reactivity to parvalbumin, a 12 kd protein present in fish extracts from six different species. Reducing extraction conditions had no effect on IgE binding to parvalbumins, whereas periodate treatment and depletion of protein-bound calcium led to a substantial reduction of IgE binding. Parvalbumins from six different species contained cross-reactive IgE epitopes. CONCLUSION: Parvalbumin represents a cross-reactive fish allergen. It contains IgE epitopes that are sensitive to periodate treatment and Ca2+-depletion.
Allergens↗
Recombinant birch pollen allergens (rBet v 1 and rBet v 2) contain most of the IgE epitopes present in birch, alder, hornbeam, hazel, and oak pollen: a quantitative IgE inhibition study with sera from different populations.
BACKGROUND: Pollen from trees of the order Fagales are important allergen sources in most parts of the world. Clinical, immunochemical, and molecular biology studies indicate that they contain cross-reactive allergens. The major birch pollen allergen, Bet v 1, and birch profilin, Bet v 2, a highly cross-reactive allergen, have been cloned and expressed in Escherichia coli. OBJECTIVE: The purpose of this study was to demonstrate the presence of allergens in Fagales pollens that share IgE epitopes with recombinant Bet v 1 and Bet v 2 and to determine the percentage of birch, alder, hornbeam, hazel, and oak pollen-specific IgE that can be preabsorbed with rBet v 1 and rBet v 2 from 102 sera of different populations of subjects allergic to Fagales tree pollen. METHODS: The presence of rBet v 1- and rBet v 2-homologous allergens in tree pollen extracts was investigated by IgE immunoblot inhibition experiments, and the percentage of tree (birch, alder, hornbeam, hazel, and oak) pollen-specific IgE that was bound by a mixture of rBet v 1 and rBet v 2 was determined by RAST-based quantitative IgE inhibition experiments. The clinical significance of IgE antibody cross-reactivity was studied by skin prick testing with rBet v 1, rBet v 2, and Fagales pollen extracts. RESULTS: Natural birch, alder, hornbeam, hazel, and oak pollen contain allergens that share IgE epitopes with rBet v 1 and rBet v 2. A combination of rBet v 1 and rBet v 2 accounted for 82% of tree pollen-specific IgE on average. Most of the tree pollen-specific IgE was directed against rBet v 1. CONCLUSION: rBet v 1 and rBet v 2 contain most of the Fagales pollen-specific IgE epitopes and may therefore substitute natural tree pollen extracts not only for diagnosis but also for patient-tailored immunotherapy of tree pollen allergy.
Adult↗
Identification of common allergenic structures in mugwort and ragweed pollen.
UNLABELLED: Identification of common allergenic structures in mugwort and ragweed pollen. BACKGROUND: Despite the rare occurrence of ragweed in Middle Europe, a surprisingly high number of patients allergic to mugwort, a frequently encountered weed, display IgE reactivity against ragweed pollen allergens. OBJECTIVE: The aim of this study was to investigate whether the high prevalence of IgE reactivity against ragweed in patients allergic to mugwort is caused by the presence of common allergenic determinants. We also sought to characterize any cross-reactive allergens. METHODS: Common allergenic structures in mugwort and ragweed pollen were characterized by qualitative IgE immunoblot inhibition experiments performed with natural allergen extracts and recombinant allergens. The degree of cross-reactivity was estimated by quantitative CAP-FEIA competitions. The clinical significance of cross-reactive IgE antibodies was studied with histamine release experiments and nasal provocation tests. RESULTS: Mugwort and ragweed RAST values were significantly correlated in a population of 82 Austrian patients allergic to mugwort. IgE antibodies cross-reacted with allergens of comparable molecular weight that were present in both extracts. By using recombinant birch profilin and specific antisera for IgE inhibition experiments, profilin was identified as one of the cross-reactive components in mugwort and ragweed pollen. Preincubation of sera from patients allergic to mugwort with mugwort extract inhibited IgE binding to ragweed pollen extract greater than 80%. Mugwort and ragweed pollen extract induced comparable histamine release and reduction of nasal air flow in a patient with IgE reactivity against the major mugwort allergen Art v 1. CONCLUSION: In addition to profilin, mugwort and ragweed pollen contain a number of cross-reactive allergens, among them the major mugwort allergen Art v 1. Cross-reactive IgE antibodies can lead to clinically significant allergic reactions.
Allergens↗
In situ localization of a high molecular weight cross-reactive allergen in pollen and plant-derived food by immunogold electron microscopy.
BACKGROUND: A high molecular weight (60 kd) allergen has been recently identified as a cross-reactive moiety in pollen and plant-derived food. While the cross-reactive allergen has been characterized by immunochemical techniques, little is known concerning its biologic properties. OBJECTIVE: In this investigation we studied the in situ localization of the 60 kd cross-reactive allergen in tree, grass, and weed pollen, as well as in plant-derived food (apple and celery). METHODS: A monoclonal antibody (3A4) that was raised against the major mugwort pollen allergen, Art v 1, was used to demonstrate the presence of related allergens in nitrocellulose-blotted pollen and plant-food extracts. The tissue localization of the cross-reactive allergen was investigated by immunogold electron microscopy. RESULTS: Monoclonal antibody 3A4 recognized IgE epitopes of the 60 kd mugwort allergen and cross-reacted with moieties of comparable molecular weights in birch and timothy grass pollen, as well as in apple and celery extracts. In pollen and plant-derived food the allergen could be localized intracellularly in ribosome-rich areas in the mitochondria and the nucleus. No labeling was observed in the pollen or cell walls or in organelles that are engaged in storage (e.g., starch granules and lipid particles). CONCLUSION: Tree, grass, and weed pollen, as well as plant-derived foods, contain a high molecular weight Art v 1-cross-reactive allergen that maps to similar cell compartments.
Allergens↗
IgE antibodies to recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) account for a high percentage of grass pollen-specific IgE.
BACKGROUND: Pollen from different grass species are some of the most potent elicitors of Type I allergy worldwide. The characterization of antigenic structures and IgE epitopes common to different grass species is relevant to define reagents for diagnosis and specific therapy of grass pollen allergy. OBJECTIVE: The purpose of this study was to estimate the percentage of IgE directed to common, cross-reactive, or both types of epitopes shared by recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) and natural pollen extracts from nine different monocots (Anthoxanthum odoratum, Avena sativa, Cynodon dactylon, Lolium perenne, Phragmites australis, Poa pratensis, Secale cereale, Triticum sativum, Zea mays) by using sera from different populations. METHODS: Natural pollen extracts from nine different monocot species were characterized regarding their allergen contents by using specific antibodies and by IgE immunoblot inhibition with recombinant allergens. The percentage of grass pollen-specific IgE that was preabsorbed with a combination of recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) and recombinant birch profilin (Bet v 2) was determined by ELISA in sera from 193 European, American, and Asian subjects. RESULTS: IgE to recombinant pollen allergens accounted for a mean 59% of grass pollen-specific IgE. A lower inhibition of IgE binding to certain natural extracts (C. dactylon and Z. mays) could be attributed to the absence of immunologically detectable group 5 and group 2 allergens in these species. CONCLUSION: We define four recombinant pollen allergens that account for a substantial proportion of grass pollen-specific IgE. The recombinant pollen allergens characterized may represent candidates not only for diagnosis but also for patient-tailored immunotherapy of grass pollen allergy.
Adult↗
Allergen immunotherapy: therapeutic vaccines for allergic diseases. World Health Organization. American academy of Allergy, Asthma and Immunology.
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Allergens↗
Molecular characterization of a cytokinin-inducible periwinkle protein showing sequence homology with pathogenesis-related proteins and the Bet v 1 allergen family.
Cytokinin treatment of periwinkle callus cultures increased the accumulation of a protein, designated T1, in two-dimensional separated protein extracts. The first 30 NH2-terminal amino acids were determined by Edman degradation and showed significant sequence homology with intracellular pathogenesis-related (IPR) plant proteins and the Bet v 1 allergen family. The deduced amino acid sequence of cDNAs coding for T1, isolated by RT-PCR and 5' RACE-PCR, exhibited an average sequence identity of 40% with both IPR and Bet v 1-related allergens. T1 and all related proteins contained a p-loop motif typically found in nucleotide-binding proteins as the most conserved sequence feature. Northern blot analysis showed that cytokinin treatment of periwinkle callus induced T1 transcripts, whereas addition of 2,4-dichlorophenoxyacetic acid inhibited this accumulation. Hybridization of genomic periwinkle DNA with the T1 cDNA suggested that the protein is encoded by a single-copy gene. Immunoblot studies with a panel of Bet v 1-specific antibodies and sera from Bet v 1 allergic individuals identified T1 as a protein that is immunologically distinct from the Bet v 1 allergen family and has no allergenic properties.
Allergens↗
Molecular cloning and mRNA localization of tomato pollen profilin.
The actin cytoskeleton plays an important role in the growth of pollen tube. The actin-binding protein profilin could play a role in regulating the organization of the actin filaments. Using the RT-PCR technique, we isolated a cDNA clone (designated LePro 1) encoding profilin from pollen grains of tomato (Lycopersicon esculentum Mill. cv. Moneymaker). Sequence analysis of the insert shows 87% similarity to tobacco ntPro2, 78% to timothy grass profilin, 77% to Arabidopsis AthPRF4, 77% to maize ZmPro3, and 73% to birch profilin. Both quantitative PCR and RNA gel blot analyses demonstrated that LePro 1 is expressed in a tissue- or cell-type specific manner in the tomato plant. In situ hybridization of 2 microns thick anther sections using a non-radioactive labeling method reveals that LePro 1 is expressed only in pollen grains, with undetectable transcription in other parts of the anther or the other organs. Phylogenetic analysis of amino acid sequences of 18 plant profilins indicates that two distinct profilin gene classes are present in higher plants. One is pollen-specific, another is constitutive. LePro 1 belongs to the former class.
Amino Acid Sequence↗
Molecular characterization of an autoallergen, Hom s 1, identified by serum IgE from atopic dermatitis patients.
Atopy is a genetically determined disorder that affects 10%-20% of the population. Many symptoms of patients with atopy (allergic rhinitis, conjunctivitis, asthma, and anaphylaxis) result from events occurring after crosslinking of cell-bound IgE by per se innocuous environmental antigens. The frequently raised hypothesis that autosensitization can also be a pathogenetic factor in atopy, gained support by our recent demonstration of IgE antibodies against human proteins in atopic dermatitis patients. To unravel the molecular nature of IgE-defined autoantigens, we used serum IgE from atopic dermatitis patients to screen a human epithelial cDNA expression library. One of the cDNA-encoding IgE-reactive products contained 1501 bp of a 2274 bp open-reading frame finally identified by sequence analysis of two additional cDNA clones resulting from oligonucleotide screening. The IgE-defined autoantigen, designated Hom s 1, exhibited an almost complete sequence identity with a recently described antigen recognized by cytotoxic T cells of a squamous cell carcinoma patient. Purified recombinant Hom s 1 specifically bound IgE from patients with severe atopy. When used as immunogen in rabbits, recombinant Hom s 1 gave rise to an anti-serum that reacted with a cytoplasmic protein exhibiting a broad cellular and tissue reactivity (skin, lung >> gastrointestinal tract >> muscle, brain) and identified a 55 kDa protein in blotted serum IgE preparations. The attractive possibility remains that the Hom s 1-triggered IgE response contributes to the events resulting in allergic tissue inflammation. If so, the respective recombinant molecule may serve as a paradigmatic tool for the diagnosis and treatment of patients with "intrinsic" atopy.
Allergens↗
Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.
Recently we demonstrated that a high percentage of atopic dermatitis (AD) patients displayed specific immunoglobulin E reactivity to human proteins. Here we show that IgE autoreactivity is found predominantly in AD patients with severe skin manifestations and reveal the molecular nature of four IgE autoantigens. An expression cDNA library constructed from a human epithelial cell line (A 431) was screened with serum IgE from two AD patients. DNA sequence analysis of three IgE-reactive clones identified the alpha-chain of the nascent polypeptide-associated complex, cytokeratin type II, and the BCL7B oncogen as atopy-related IgE autoantigens (ara). The fourth cDNA coded for an IgE autoantigen containing a typical calcium binding motif that occurred in histogenetically different cells and tissues (keratinocytes, muscle, brain). Recombinant Escherichia coli-expressed IgE autoantigens bound IgE from AD but not from patients with other immunologically mediated disorders (graft vs. host disease, systemic lupus erythematosus) and elicited immediate type skin reactions in AD patients. In serum samples collected from an AD patient over a period of 5 years, IgE anti-ara NAC antibody levels peaked during disease exacerbation. Our finding that ara BCL7B was detected in serum bound to IgE antibodies suggests that intracellular IgE autoantigens can become released after tissue damage and may occur as IgE immune complexes. Via binding to antigen presenting cells as well as to effector cells, IgE autoantigen immune complexes may contribute to exacerbation and/or perpetuation of severe atopic diseases even in the absence of exogenous allergens.
Amino Acid Sequence↗
Recombinant allergens.
A great variety of recombinant plant, mite, mold, mammal, and insect allergens have been expressed in heterologous hosts (e.g., Escherichia coli), their cDNA being used as a template. The number of biologically active recombinant allergens available for experimental, diagnostic, and therapeutic purposes is increasing tremendously. Recombinant allergens have proven to be valuable tools to investigate T-cell and B-cell recognition of allergens as well as to study mechanisms of specific IgE regulation. The immunologic equivalence of many relevant recombinant allergens with their natural counterparts has been demonstrated, and the three-dimensional structures of several recombinant allergens have been described recently. As a result of extensive cross-reactivities among the relevant allergens, it appears that the number of epitopes needed for diagnosis and specific immunotherapy is less diverse than originally anticipated and might be soon covered by recombinant molecules. Recombinant allergens have been used for successful in vitro, as well as in vivo, allergy diagnosis, and work is in progress to produce recombinant allergen derivatives with reduced anaphylactic potential to improve current forms of immunotherapy.
Allergens↗
Recombinant allergens: the future of the diagnosis and treatment of atopic allergy.
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Allergens↗