D. Lüttkopf

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.

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.

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.

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.

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.

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.

Optimized allergen extracts and recombinant allergens in diagnostic applications.

Due to inherent enzyme activities and other factors, allergen extracts from fruits, vegetables and other plant foods often lack sufficient biological activity (1–4). This lability also leads to consistency problems with different lots of commercial extracts, or to large variations between extracts from different manufacturers. In certain cases, special extraction procedures are required to obtain extracts that are suitable for in vitro an in vivo diagnosis (4–6). Although this problem has been known for more than 20 years (7), this article will show that some problems still need to be solved. In general, allergen extracts from plant materials contain many glycoproteins with N-linked glycans that bind IgE in a subgroup of patients sensitized to pollen and food. Some authors think that these IgE responses are clinically irrelevant (8, 9), whereas others have shown that IgE responses specific for these so-called crossreactive carbohydrate determinants (CCD) are frequently present in patients with adverse reactions to certain foods, and can be the unique detectable IgE response to these foods in such patients (10–12). Whether or not anti-glycan IgE is able to trigger allergic symptoms still needs to be answered. However, specific IgE responses in allergic patients are undoubtedly linked to the pathomechanism of the disease and should be taken into consideration. Diagnostic tools to pick up anti-CCD antibodies have been developed (10, 11) and are useful to highlight whether IgE reactivity to an allergen extract is caused by protein allergens or by carbohydrate moieties. In this context, recombinant food allergens produced in bacteria that completely lack N-glycosylations may not only be useful to capture anti-protein IgE responses, but also to proceed from an extract-specific diagnosis to a component-resolved diagnosis (13). This has been achieved with inhalant allergens, including complex systems such as grass pollen (14), and initial studies have been published for foods such as peanut (15) and pear (13). Componentresolved diagnosis may also offer the opportunity to distinguish between IgE directed against ‘‘mild’’ and ‘‘severe’’ food allergens (9). In principle, the panel of recombinant or purified natural allergens used to replace the extract should be as complete as possible, and the recombinant compounds should closely resemble the IgE reactivity of their natural homologues. Here we report examples selected from the area of pollenrelated food allergy that address the potential of optimized allergen extracts and recombinant allergens in the determination of food-specific IgE antibodies.

Regulatory environment for allergen‐specific immunotherapy

To cite this article: Kaul S, May S, Lüttkopf D, Vieths S. Regulatory environment for allergen‐specific immunotherapy. Allergy 2011; 66: 753–764.

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.

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’.