Kay Fötisch

The CREATE Project : development of certified reference materials for allergenic products and validation of methods for their quantification

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE‐binding potencies as their focus. Unfortunately, each company is using their own in‐house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

N- and O-linked oligosaccharides of allergenic glycoproteins.

Cross-linking of cell-bound IgE on mast cells or basophils by polyvalent antigens causes the release of histamine and other mediators of the allergic response which then lead to the development of allergic symptoms. In this event not only peptide epitopes, but also carbohydrates can act as cross-linking elements. Since peptide epitopes of allergens are subject of most published studies, this review is focused on glycosidic epitopes. The current knowledge of the structures and possible epitopes of oligosaccharides linked to allergenic glycoproteins is briefly reviewed, showing that complex plant N-glycans containing α1,3 fucose and β1,2 xylose are most frequently involved in the structures of IgE epitopes. In own studies a prevalence of up to 29% anti-glycan IgE was determined among pollen-allergic patients. The clinical relevance of these carbohydrate specific IgE antibodies is still a matter of controversial discussions.

Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure.

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.

Component-resolved in vitro diagnosis in carrot allergy: does the use of recombinant carrot allergens improve the reliability of the diagnostic procedure?

Background In Europe, pollen‐related food allergy is the most frequent form of food allergy in adults. Reliability of current diagnostic procedures, however, is poor and therapeutic options are not available.

Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen

BACKGROUND Allergen-specific immunotherapy for food allergies, including peach allergy, has not been established. Use of allergens with reduced allergenic potential and preserved immunogenicity could improve the safety and efficacy of allergen-specific immunotherapy. OBJECTIVE We sought to create a hypoallergenic derivative of the major peach allergen Pru p 3 and to characterize its biochemical and immunologic properties. METHODS A Pru p 3 folding variant generated by means of reduction and alkylation was investigated for structural integrity and stability to gastrointestinal enzymes. IgE reactivity and allergenic potency were determined by means of immunoblotting, ELISA, and in vitro mediator release assay with sera from patients with peach allergy. T-cell immunogenicity was investigated by using human allergen-specific T cells and CBA/J mice immunized with either native Pru p 3 (nPru p 3) or reduced and alkylated (R/A) Pru p 3. Pru p 3 processing by endolysosomal fractions of dendritic cells and antigenicity was examined in mice. RESULTS Unfolding of Pru p 3 reduced its high resistance to gastrointestinal proteolysis and almost completely abrogated its IgE reactivity and allergenic potency. However, R/A Pru p 3 was capable of stimulating human and murine T cells. Endolysosomal degradation of R/A Pru p 3 was accelerated in comparison with nPru p 3, but similar peptides were generated. IgG and IgE antibodies raised against nPru p 3 showed almost no cross-reactivity with R/A Pru p 3. Moreover, the antigenicity of R/A Pru p 3 was strongly reduced. CONCLUSION Unfolded Pru p 3 showed reduced allergenicity and antigenicity and preserved T-cell immunogenicity. The hypoallergenic variant of Pru p 3 could be a promising vaccine candidate for specific immunotherapy of peach allergy.

IgE antibodies specific for carbohydrates in a patient allergic to gum arabic (Acacia senegal)

The present study deals with the detailed investigation of the IgE antibody response of a gum arabic‐allergic patient. The patient showed multiple serologic and skin test sensitizations to a range of pollen, other inhalants and foods, and bee venom, and to the recombinant allergens Bet v 1 and Bet v 2. Moreover, the patients serum reacted strongly to gum‐arabic extract. The NaIO4‐treated and thus deglycosylated extract showed no binding to IgE. In contrast, removal of the protein backbone by basic hydrolysis did not deplete the IgE reactivity. Therefore, it is concluded that the gum arabic‐specific IgE antibodies of this patient were mainly directed against the carbohydrate fraction of this material. In IgE‐inhibition assays, cross‐reactions occurred in the range of 60% between gum arabic and known immunogenic A‐glycans containing o(l‐3‐linked fucose. Since the inhibition graphs were not parallel and the inhibition was not complete with heterologue antigens, the cross‐reacting epitopes of gum arabic appeared to be different from the latter well‐known cross‐reactive carbohydrate determinants (CCD). Inhibition may have been caused by a partial immunologic identity of the investigated carbohydrate moieties. A strong IgE response to the fucose‐containing glycan from bromelain was measured in a glycan ELISA that utilizes purified glycopeptides at the solid phase. This response, which may explain the multiple sensitizations without clinical significance diagnosed in the patient, could originate from inhalation of pollen, which is known to contain similar glycans, or from occupational sensitization during work as a baker and confectioner. Since the gum‐arabic protein showed only very weak participation in the IgE reactivity, the clinical symptoms of the patient caused by gum arabic may be attributed to carbohydrate epitopes. Due to the repetitive polysaccharide sequence of gum arabic, several epitopes for the cross‐linking of IgE should exist.

Pichia pastoris is superior to E. coli for the production of recombinant allergenic non-specific lipid-transfer proteins.

Non-specific lipid-transfer proteins (nsLTP) from food and pollen are clinically important allergens, especially in patients recruited from the Mediterranean area. For the use of recombinant nsLTPs in allergy diagnosis and preclinical allergy studies the preparation of nsLTPs in a properly folded and biologically active form is required. Using hazelnut nsLTP (Cor a 8) as a model allergen, heterologous over-expression in Escherichia coli and Pichia pastoris was compared. Recombinant Cor a 8 derived from E. coli and P. pastoris was purified by IMAC and SEC or ammonium sulphate precipitation followed by IEC and SEC, respectively. The recombinant proteins were characterized with regard to IgE-binding by immunoblotting and ELISA, structure by N-terminal sequencing, CD-spectroscopy and LS and to their biological activity using an in vitro basophil histamine release assay. Purification of hazelnut nsLTP from bacterial lysate under native conditions resulted in a low yield of Cor a 8. In addition, the preparation contained non-IgE-reactive aggregations besides the IgE-reactive monomer. In contrast, the yield of rCor a 8 produced in P. pastoris was approximately 270-fold higher and impurities with oligomers have not been detected. Purified monomeric Cor a 8 from bacteria and yeast showed similar IgE-antibody reactivity and secondary structures, and both were capable of inducing histamine release from basophils. In summary, P. pastoris is superior to E. coli as expression system for the production of large quantities of soluble, properly folded, and biologically active rCor a 8.

Orange-induced skin lesions in patients with atopic eczema: evidence for a non-IgE-mediated mechanism.

Oranges are suspected of inducing adverse skin reactions in patients with atopic eczema. We studied 21 adult patients with atopic eczema and a history of adverse reactions to oranges and 10 patients without. A dietary history, skin tests, serum IgE and oral provocation tests with oranges were obtained. Severity of eczema was monitored by SCORAD, and serum tryptase, eosinophil cationic protein and urinary methylhistamine were measured. No allergic reactions were found to orange in skin prick or patch tests. However, 23 patients (74%) had specific serum IgE to orange. Oral provocation testing resulted in pruritic eczematous or maculopapular skin lesions predominantly at the predilection sites in 16 patients (52%). The SCORAD increased significantly in patients positive to the oral provocation test (p <0.05). Specific IgE to orange did not correlate with the clinical outcome of the oral provocation test. No significant changes were found in serum mast cell tryptase, eosinophil cationic protein or in urinary methylhistamine excretion. The negative results in the skin tests and a lack of correlation between specific IgE and oral provocation tests indicate that non-IgE-mediated mechanisms are involved in cutaneous adverse reactions to oranges in patients with atopic eczema.

Identification of a Dau c PRPlike protein (Dau c 1.03) as a new allergenic isoform in carrots (cultivar Rodelika)

Up to 25% of food allergic subjects in central Europe suffer from carrot allergy. Until now, two isoforms of the major carrot (Daucus carota) allergen Dau c 1 have been described: Dau c 1.01, comprising five variants (Dau c 1.0101–Dau c 1.0105) and Dau c 1.02.

IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen

Allergen‐specific immunotherapy (AIT) with birch pollen generates Bet v 1‐specific immunoglobulin (Ig)G4 which blocks IgE‐mediated hypersensitivity mechanisms. Whether IgG4 specific for Bet v 1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG4 antibodies are developed is under debate.