Regulatory environment for allergen-specific immunotherapy.

Products for specific immunotherapy (SIT) are medicinal products according to the European Regulations. To obtain a marketing authorization (MA) within the European Community, the quality, safety and efficacy have to be proven. During the development phase of a medicinal product, applicants have the opportunity to apply for scientific advice by national competent authorities or the European Medicines Agency (EMA) to compile a suitable development plan for the examination of quality and performance of nonclinical and clinical trials. Moreover, a paediatric investigation plan has to be submitted to the Paediatric Committee of the EMA and has to be approved before submission of an application for MA. Several regulatory procedures exist for obtaining a MA in the European Community. The national procedure leads only to marketability in one country whereas the Mutual Recognition, the Decentralized and Centralized Procedures (CP) are intended for MA in several or all member states of the European Union. The CP is mandatory for certain medicinal products, for example for drug substances derived by biotechnological processes such as recombinant allergens. Named Patient Products for SIT are a specialty because they are manufactured on the basis of an individual prescription and marketed without a MA.

The principle of homologous groups in regulatory affairs of allergen products--a proposal.

Among other legal regulations, the Note for Guidance on Allergen Products CPMP/BWP/243/96 released by the European Medicines Agency provides regulatory instructions regarding the quality of allergen extracts for diagnostic or therapeutic purposes. The current revision of this guideline intends to transform the so-called 'principle of taxonomic families' to the 'principle of homologous groups'. According to this concept, the data of one allergen extract demonstrating stability, efficacy and safety can, to a limited extent, be extrapolated to other allergen extracts belonging to the same homologous groups. The present work proposes the formation of homologous groups for pollen species and animal-derived materials on the basis of similar biochemical composition and homology/cross-reactivity of allergens or allergen sources. Some tree pollen species could be assigned to three different homologous groups, some weed pollen species to one homologous group and numerous grass pollen species to one homologous group on condition that data rely on single defined representative species. A homologous group for mites is limited to the Dermatophagoides species and the grouping of vertebrate-derived materials such as dander could be possible under restrictions. The criteria for the formation of the proposed homologous groups are illustrated in detail to provide an opportunity for extending existing homologous groups by further species in case of new insights in allergens and cross-reactivity of allergen sources. In this way, the concept of homologous groups could serve as a dynamic tool in the regulation of allergen products.

The regulatory system in europe with special emphasis on allergen products.

For each medicinal product quality, safety and efficacy have to be proven to obtain a marketing authorisation. The national competent health authorities and the European Medicines Agency (EMEA) with support of the Heads of Medicines Agencies (HMA) work together to grant marketing authorisations for medicinal products. Several regulatory procedures to apply for a marketing authorisation in the European Community (EC) and associated countries exist. After approval by a national procedure a medicinal product can be marketed in only one country. If a medicinal product should enter the markets of two or more European countries of choice the application has to undergo the Mutual Recognition Procedure (MRP) or the Decentralised Procedure (DCP). A marketing authorisation granted by the Centralised Procedure (CP) is valid in the whole EC. The CP is mandatory for certain medicinal products, for example all products derived from recombinant DNA technology including recombinant allergens. The guidance documents applicable to allergen products comprise general as well as product-specific guidelines such as the Note for Guidance on Allergen Products and the Monograph on Allergen Products of the European Pharmacopoeia. So-called 'named patient products' have a special status and are applied to patients without having a marketing authorisation. Recombinant allergens as medicinal products are insufficiently covered by the existing allergen product-specific guidelines, but product-specific guidelines are in the development stage.

Mediator release assays based on human or murine immunoglobulin E in allergen standardization.

BACKGROUND: The biological potency of allergens can be measured by provoking mediator release from effector cells. As established immunochemical methods in allergen standardization only determine inhibition potency or major allergen content, routine tests for biological potency may enhance standardization and batch control of allergen products. OBJECTIVE: The general performance and application potential of biological in vitro assays in batch control and standardization of allergens and as a tool for verifying activity and stability of allergen standards were analysed. METHODS: Allergen extracts of five clinically relevant allergens from three to five different manufacturers were investigated. A CAP-IgE-inhibition assay was compared with mediator release assay (MRA)s based on murine or human basophils. Rat basophilic leukaemia (RBL) cells were passively sensitized with pooled murine allergen-specific IgE-containing sera. Humanized RBL cells and human-stripped basophils were sensitized with pooled patient's sera, which were also used for the CAP-IgE-inhibition assay. Allergen specificity of the sera was determined by immunoblotting. RESULTS: A good batch-to-batch consistency was found with each assay among all manufacturers and allergens tested. Between different manufacturers, the products showed differences in activity and the various assays indicated an almost identical ranking. However, the biological assays revealed qualitative differences of biological activity or composition of allergen preparations undetectable by IgE-inhibition assay. CONCLUSIONS: MRAs provide refined information on allergen activity, either confirming the results of IgE-inhibition assay, or indicating differences requiring further investigation, and represent a highly sensitive novel tool in allergen standardization. By using permanently cultivated cell lines, repeated venepuncture to obtain human basophils is avoided. As in the RBL assay, the coefficient of variation for the release values were below 15% and for the ED50 below 25%, the assay is suitable to determine differences that are relevant for batch control purposes.

Structural, immunological and functional properties of natural recombinant Pen a 1, the major allergen of Brown Shrimp, Penaeus aztecus.

BACKGROUND: Recombinant allergens are considered the basis for new diagnostic approaches and development of novel strategies of allergen-specific immunotherapy. As Pen a 1 from brown shrimp Penaeus aztecus is the only major allergen of shrimp and binds up to 75% of all shrimp-specific IgE antibodies this molecule may be an excellent model for the usage of allergens with reduced IgE antibody-binding capacity for specific immunotherapy. AIM: The aim was to clone, express and characterize a full-length recombinant Pen a 1 molecule and compare it with natural Pen a 1 in regard to structural and immunological parameters such as IgE antibody capacity and ability to induce IgE-mediated mediator release. METHODS: Total RNA was isolated from P. aztecus and a rapid amplification of cDNA ends (5' RACE) was performed to obtain full-length cDNA coding for Pen a 1. Using a gene-specific primer, PCR was performed and full-length cDNA was cloned and sequenced. Recombinant His-tagged Pen a 1 was isolated from Escherichia coli under native conditions by immobilized metal affinity chromatography. Secondary structure of natural and recombinant Pen a 1 was compared by circular dichroism (CD) spectroscopy, and the IgE antibody-binding capacity evaluated by RAST. The allergenic potency was tested by the capability of natural and recombinant Pen a 1 to induce mediator release in a murine and human in vitro model of IgE-mediated type I allergy. RESULTS: The deduced amino-acid sequence was 284 residues long and amino-acid sequence identities with allergenic and non-allergenic tropomyosins ranged from 80% to 99% and 51% to 58%, respectively. The analysis of the secondary structure of natural and recombinant Pen a 1 by CD spectroscopic analysis showed that both nPen a 1 and rPen a 1 had alpha-helical conformation that is typical for tropomyosin. The IgE antibody binding capacities of nPen a 1 and r Pen a1 were found to be essentially identical by RAST. The mediator release experiments using both wild-type and humanized rat basophilic leukaemia 30/25 cells showed that rPen a 1 and nPen a 1 induced a similar level of mast cell activation. CONCLUSIONS: Recombinant Pen a 1 and natural Pen a 1 are structurally and immunologically identical and rPen a 1 may be used as the basis for component-resolved diagnosis and the generation of modified shrimp tropomyosin for allergen-specific immunotherapy. The results of the animal studies indicate that C3H/HeJ mice that were sensitized with shrimp extract in combination with cholera toxin as adjuvant may be a suitable model to study shrimp allergy.

Characterization of the T cell response to the major hazelnut allergen, Cor a 1.04: evidence for a relevant T cell epitope not cross-reactive with homologous pollen allergens.

BACKGROUND: IgE antibodies specific for the major birch-pollen allergen, Bet v 1, cross-react with homologous allergens in particular foods, e.g. apples, carrots and hazelnuts. In a high number of tree pollen-allergic individuals, this cross-reactivity causes clinical symptoms, commonly known as the 'birch-fruit-syndrome'. OBJECTIVE: To characterize the T cell response to the Bet v 1-related major allergen in hazelnuts, Cor a 1.04, and its cellular cross-reactivity with Bet v 1 and the homologous hazel pollen allergen, Cor a 1. METHODS: Using recombinant Cor a 1.04, T cell lines (TCL) and T cell clones (TCC) were established from peripheral blood mononuclear cells of tree pollen-allergic patients with associated food allergy. T cell epitopes were determined using overlapping synthetic peptides in Cor a 1.04-reactive TCL and TCC. In parallel, reactivity to Bet v 1 and Cor a 1 was tested. RESULTS: In total, 20 distinct T cell epitopes on the hazelnut allergen were identified. Several Cor a 1.04-specific TCL and TCC reacted with pollen allergens albeit less pronounced than with the hazelnut allergen. Several Cor a 1.04-specific TCC did not react with pollen allergens. Interestingly, these clones were found to react with the Bet v 1-related major allergen in carrots, Dau c 1. The cellular cross-reactivity between both food allergens could be associated with the most frequently recognized T cell epitope of Cor a 1.04, Cor a 1.04(142-153). CONCLUSIONS: The major hazelnut allergen cross-reacts with the major allergens of birch and hazel pollen but apparently contains a relevant T cell epitope not shared with pollen allergens. Our finding may have important implications for the specific immunotherapy of tree pollen-allergic patients suffering from concomitant hazelnut allergy.

Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system.

BACKGROUND: Biochemical and immunochemical methods used for batch control of allergen extracts rely on the binding of IgE molecules to allergens. They do not measure the ability of a protein to induce type I allergic reactions. Therefore, a biological assay was established that is based on the cellular mechanisms of allergies in order to assess the cross-linking capacity of allergens. METHODS: Rat basophilic leukaemia cells were transfected with cDNA coding for the human high affinity IgE receptor chains. The surface expression of the IgE-binding alpha-chain was detected by FACS analysis and the functional integration of the 'humanized' receptors into the signal transduction cascade was addressed by intracellular calcium mobilization. Mediator release was measured in response to human IgE and a variety of cross-linking allergen preparations. RESULTS: Several clones were obtained that were able to bind allergen-specific human IgE. The results of the biological assay were compared with those obtained by immunochemical methods. The biological assay was used to determine the potency of allergen extracts, including highly diluted products that cannot be analysed by conventional methods. CONCLUSION: A stable 'humanized' basophil cell line was established that will be a useful tool for the standardization and batch control of allergen extracts. Because of its high sensitivity, it can also be used to detect minute quantities of potentially allergenic proteins, e.g. in processed foods. In addition, the test may support the development of novel allergy vaccines, such as recombinant hypoallergenic molecules.

Roasted hazelnuts--allergenic activity evaluated by double-blind, placebo-controlled food challenge.

BACKGROUND: Allergy to hazelnuts is a common example of birch pollen related food allergy. Symptoms upon ingestion are often confined to the mouth and throat, but severe systemic reactions have been described in some patients. The aim of the study was to evaluate the reduction in allergenicity by roasting of the nuts. METHODS: Double-blind, placebo-controlled food challenges (DBPCFC) with roasted hazelnuts (140 degrees C, 40 min) were performed in 17 birch pollen allergic patients with DBPCFC-confirmed food allergy to raw hazelnuts. The effect of roasting was further evaluated by skin prick test (SPT), histamine release (HR), measurement of specific IgE, and IgE-inhibition experiments. RESULTS: In 5/17 patients the DBPCFC with the roasted nuts were positive. The symptoms were generally mild and included OAS (oral allergy syndrome) in all patients. Roasting of the nuts significantly reduced the allergenic activity evaluated by SPT, HR, specific IgE, and IgE-inhibition. Immunoblotting experiments with recombinant hazelnut allergens showed sensitization against Cor a 1.04 in 16/17 patients and against Cor a 2 in 7/17 patients. None of the patients were sensitized to Cor a 8. Challenge-positive patients did not differ from the rest in IgE-binding pattern. CONCLUSIONS: All the applied methods indicated that roasting of hazelnuts reduces the allergenicity, but since 5/17 birch pollen allergic patients were DBPCFC-positive to the roasted nuts, ingestion of roasted hazelnuts or products containing roasted hazelnuts can not be considered safe for a number of hazelnut allergic consumers. For patients with a history of severe allergic symptoms upon ingestion of hazelnuts, thorough and conscientious food labelling of hazelnuts and hazelnut residues is essential.

Allergens in celery and zucchini.

The aim of this study was to confirm allergy to celery tuber and to zucchini, for the first time, by DBPCFC, and to identify the allergens recognized by IgE from DBPCFC-positive patients. Therefore, raw vegetables were hidden in a broccoli drink, and a DBPCFC-procedure was developed that consisted of a spit and swallow protocol, making sure that the procedure was safe for the patients and that reactions strictly localized to the oral cavity as well as systemic reactions could be reproduced by DBPCFC. The allergens in celery and zucchini extract were identified by immunoblot inhibition using allergen extracts, recombinant allergens and purified N-glycans as inhibitors. Celery allergy was confirmed in 69% (22/32) of subjects with a positive case history. Four subjects with a history of allergic reactions to zucchini had a positive DBPCFC to this vegetable. During DBPCFC, systemic reactions were provoked in 50% (11/22) of the patients to celery, and in 3/4 of the zucchini-allergic patients. The Bet v 1-related major celery allergen was detected by IgE of 59% (13/22) of the patients. Cross-reactive carbohydrate epitopes (CCD) bound IgE of 55% (12/22) of the celery-allergic patients and in 2/4 of the subjects with zucchini allergy. Profilin was a food allergen in celery in 23% (5/22) and in zucchini in 2/4 of the cases. A zucchini-specific allergen was detected by IgE from one patient. We conclude that ubiquitous cross-reactive structures are important in allergy to both, celery and zucchini, and that a specific association to birch pollen allergy exists in allergy to celery (mediated by Api g 1), but not in zucchini allergy.

Influence of food processing on the allergenicity of celery: DBPCFC with celery spice and cooked celery in patients with celery allergy.

BACKGROUND: Celery root is often consumed in a processed form as a cooked vegetable or as a spice. So far, however, there has been no information about the allergenicity of processed celery in celery-allergic patients. METHODS: In 12 patients with a history of allergic reactions to raw or raw and cooked celery, double-blind placebo-controlled food challenges (DBPCFCs) with raw celery (n = 10), cooked celery (110 degrees C/15 min; n = 11), and celery spice (n = 5) were performed. Nine patients underwent an open mucosal challenge with four samples of canned celery retorted at Co-values (cooking effect) of 7.45-76.07 (corresponding to the time periods in minutes at a thermal influence of 100 degrees C). IgE immunoblot analysis of celery extract was performed with sera of all challenged patients. The thermal stability of celery allergen was investigated by enzyme allergosorbent test (EAST) inhibition. Furthermore, intraperitoneal immunization of mice followed by a rat basophil leukemia (RBL) cell mediator release assay was used as a biological in vitro model to assess the allergenicity of processed celery. RESULTS: Six out of 11 patients showed a positive DBPCFC to cooked celery and five out of five patients to celery spice. Allergenicity of celery was preserved in four patients with a positive DBPCFC to cooked celery even if celery was treated at a Co-value of 76.07. Patients with positive DBPCFC to cooked celery reacted to known celery allergens (Api g 1, Api g 4, cross-reactive carbohydrate determinants CCD). EAST inhibition showed that heat resistance of celery allergens decreases in the following order: CCD > Api g 4 > Api g 1. Accordingly, five of six patients with a positive DBPCFC to cooked celery were sensitized to profilin and/or CCD. The murine model reflected the reactivity of patients sensitized to the major allergen Api g 1. CONCLUSIONS: 1) In a subset of patients with a positive DBPCFC to cooked celery, celery remains allergenic even after extended thermal treatment (76.07 min/100 degrees C). 2) Celery spice is allergenic for patients with an allergy to raw celery. 3) RBL cells sensitized with mouse IgE to raw celery may serve as a useful tool for screening the potential allergenicity of heat-processed products containing celery.

Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01.

The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1. IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant). Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404. It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen. Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.

IgE binding to unique hazelnut allergens: identification of non pollen-related and heat-stable hazelnut allergens eliciting severe allergic reactions.

BACKGROUND: Usually hazelnut allergic patients suffer from the tree pollen associated oral allergy syndrome (OAS) caused by cross-reactive structures. Anaphylactic reactions elicited by hazelnuts happen rarely but are of high clinical significance. Considering that hazelnuts are ingredients in processed foods, hazelnuts may play an important role as hidden allergens for these high risk patients. Therefore, we analyzed the IgE reactivity of a young woman with severe allergic reactions after ingestion of hazelnuts without any association to tree pollen allergy. AIM OF THE STUDY: The aim of this study was to identify and characterize these potent hazelnut-specific allergens. We compared these allergens to structures displayed by sera from patients with a completely or partially non pollen-related hazelnut allergy and with birch pollen-related hazelnut allergy. None of the sera had a clinical history of anaphylaxis. Special emphasis was placed on the heat stability and cross-reactivity of these allergens. METHODS/RESULTS: Using Western blotting with extract from birch pollen and EAST inhibition techniques we were able to show that the allergens in the serum sample of the young woman were not cross-reactive with birch pollen. Immunoblot experiments with extracts from native and heated hazelnuts and EAST inhibition tests further characterized these allergens to be heat-stable. Unlike the IgE binding pattern of the sera from the patients with pollen-related hazelnut allergy, low molecular weight proteins below 10 kDa were identified by the sera from the patients without pollinosis. CONCLUSIONS: Since the binding pattern of the serum sample of the young woman was different from that of the sera from patients without pollen allergy but less severe symptoms, we assume an association between single non pollen-dependent hazelnut allergens in the low molecular range and severe allergic reactions. These results enable us to approach a subgroup of hazelnut allergens which we believe to be responsible for anaphylactic reactions in hazelnut allergic patients after ingestion of heat-stable hazelnut structures in processed food stuff, independent of pollinosis.

Celery allergy confirmed by double-blind, placebo-controlled food challenge: a clinical study in 32 subjects with a history of adverse reactions to celery root.

BACKGROUND: Celery root is a frequent cause of food allergy in pollen-sensitized patients. Because of problems in blinding challenges with fresh vegetables and the risk of anaphylactic reactions, no double-blind, placebo-controlled, food challenges (DBPCFCs) with celery have been published so far. OBJECTIVE: The aim of the study was to confirm the clinical relevance of celery as a food allergen by DBPCFCs and to evaluate current diagnostic procedures in patients with true allergy. METHODS: DBPCFCs were performed in 32 patients with a history of an allergic reaction to celery. The patients underwent skin prick tests (SPTs) with celery extracts, crude celery, and different pollen extracts. Specific IgE for celery was determined by using the CAP method. RESULTS: Twenty-two of 32 patients had a positive DBPCFC result. Two patients reacted to placebo, and 8 patients did not respond to the challenge. Of the nonresponders, 4 reacted to an open provocation with celery. The sensitivity of CAP determination for specific IgE (> or =0.7 kU/L) to celery in patients with a positive DBPCFC result was 73%, 48% to 86% for SPTs (> or =3 mm) with commercial extracts, and 96% for prick-to-prick tests with crude celery. The positive predictive value of the SPT and CAP tests was between 87% and 96%, whereas the specificity and negative predictive values were poor. CONCLUSION: This study confirms the importance of celery as a food allergen for use in DBPCFCs. The SPT and CAP methods proved to be reliable for the diagnosis of a relevant allergy to celery in regard to sensitivity and positive predictive value but not to specificity and negative predictive value.

Celery allergens in patients with positive double-blind placebo-controlled food challenge.

BACKGROUND: Recently, for the first time, allergy to celery was confirmed by double-blind placebo-controlled food challenge (DBPCFC). Api g 1, Api g 4, cross-reactive carbohydrate determinants (CCD), and a 60 kDa allergen have been described as celery allergens. OBJECTIVE: To get insights in IgE responses of patients with a positive DBPCFC to celery tuber (celeriac) compared with patients with a negative challenge test. METHODS: Specific IgE to native and heated celery tuber and to recombinant Api g 1, the major celery allergen, were determined by enzyme allergosorbent test and immunoblotting. IgE binding to Api g 1, Api g 4, and CCD was confirmed by inhibition experiments that used recombinant Api g 1, recombinant Api g 4, pure N-glycans, and extracts of celeriac, lychee fruit, and pollens of birch, mugwort, and timothy grass as inhibitors. RESULTS: Immunoblotting with sera from 22 patients with a positive DBPCFC to celeriac confirmed the presence of known allergenic structures: The major allergen Api g 1 (16 kDa) was recognized by IgE from 13 of 22 patients (59%). Another major allergen was CCD, determined by IgE reactivity in 12 of 22 patients (55%). Celery profilin, Api g 4, was recognized by IgE from 5 of 22 patients (23%). CONCLUSION: Our DBPCFC-positive patients exclusively presented IgE to known celery allergens, although the prevalences were slightly different than were previously reported. No obvious differences were found in patients with positive IgE antibody but negative challenge test. IgE binding to all 3 structures in celeriac extract was inhibited by birch pollen extract, whereas mugwort pollen extract could only inhibit IgE reactivity to Api g 4 and CCD. Inhibition experiments with a purified carbohydrate moiety clearly showed that the IgE epitope mannose-xylose-fucose-glycan (Manalpha1-6[Xylbeta1-2]Manbeta1-4GlcNAcbeta1-4[ Fucalpha1-3]GlcNAc) or a closely related structure is present in celeriac extract and is important in patients with clinical allergy to celery.