Quality control of allergen products with mass spectrometry part I: Positioning within the EU regulatory framework.

Mass spectrometry (MS) has advanced greatly and many of its applications are ready for utilization within regulatory procedures and could significantly contribute to overcome challenges in standardization of allergen products. It seems sensible to discuss MS within the regulatory framework, before addressing technical questions. While the application to purified proteins is well established from product development to manufacturer's release analytics, its application to complex products such as allergen products is still under development. It needs to be determined where it can complement or replace established methods or where MS offers limited improvement. Despite its technical appeal and versatility, currently MS is mentioned in regulatory guidelines only as one possible measurement method. For example, no specific MS method is given in the European Pharmacopoeia. We discuss applications of MS within the EU regulatory framework. This includes their advantages and disadvantages and their positioning between research, characterization, manufacturer's release analytics and official batch testing. We discuss the qualitative detection of single and multiple allergens as proof of identity, qualitative to semi-quantitative protein profiles for batch to batch consistency testing, and quantification of allergens to state mass units of allergens. MS may also facilitate standardization of allergen products, reference products and reference standards.

Allergen-specific immunotherapy and evidence: A European regulatory perspective.

Allergen immunotherapy (AIT) has been performed for 112 years. In this article we summarize regulatory standards and challenges based on scientific evidence on AIT. Most crucial and timely aspects concerning AIT are addressed from the regulatory perspective of the authors as employees of a national competent authority in Europe: (1) product specificity; (2) clinical efficacy; (3) treatment for adults and children (needs for extrapolation); (4) allergen exposure chambers; (5) biomarkers; (6) standardization; (7) real-world evidence; (8) independent official batch release (benefit and challenges); (9) harmonization on the EU level. The Paul-Ehrlich-Institut (PEI), the Federal Institute for Vaccines and Biomedicines, in Langen near Frankfurt/Main in Germany, examines and evaluates the benefits and risks of AIT products within the course of clinical development, marketing authorization, and subsequently throughout their entire life cycle to ensure high-quality, safe, and effective AIT products.

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.

Comparison of IgE-binding capacity, cross-reactivity and biological potency of allergenic non-specific lipid transfer proteins from peach, cherry and hazelnut.

BACKGROUND: Whether the observed clinical pattern of non-specific lipid transfer protein (nsLTP)-mediated food allergies is attributable to a primary sensitization by Pru p 3 from peach and subsequent cross-reactivity with Rosaceae- and non-Rosaceae-derived foods expressing homologous allergens is still unclear. OBJECTIVE: To investigate the allergenic properties of nsLTPs from Rosaceae and non-Rosaceae foods. METHODS: In peach-, cherry- or hazelnut-allergic patients, prevalence of sensitization, IgE-binding capacity, cross-reactivity and allergenic potency of Pru p 3 was compared with Pru av 3 (cherry) and Cor a 8 (hazelnut). RESULTS: Frequency of sensitization to corresponding nsLTPs was 88, 85, and 77% in peach-, hazelnut- and cherry-allergic patients, respectively. Concomitant allergic reactions to cherry and hazelnut were reported in 51 and 44% of peach-allergic patients, respectively. In contrast to cherry allergy, hazelnut allergy was not strictly associated to peach allergy. Sensitization to Cor a 8 or Pru av 3 was strongly correlated with IgE reactivity to Pru p 3, even when subjects tolerated peach. Specific IgE was highest for Rosaceae LTPs, and cross-inhibition experiments confirmed a stronger IgE-binding capacity of Pru p 3 than Cor a 8. The biological potency of Pru p 3 and Pru av 3 was similar but stronger for both nsLTPs than that of Cor a 8. CONCLUSION: Clinical cross-reactivity of food-allergic patients in the Mediterranean area is likely attributed to a primary sensitization to Pru p 3 and serological cross-reactivity with homologous food nsLTPs. In comparison to Cor a 8, Rosaceae nsLTPs showed a stronger IgE-binding capacity and allergenic potency indicating a different epitope pattern.

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.

Characterization of the allergic T-cell response to Pru p 3, the nonspecific lipid transfer protein in peach.

BACKGROUND: Pru p 3, the nonspecific lipid transfer protein from peach, is an important plant food allergen that frequently induces systemic reactions. OBJECTIVE: We sought to analyze the allergic T-cell response to Pru p 3. METHODS: PBMCs from Italian and Spanish patients with peach allergy were stimulated with purified natural Pru p 3. Allergen-specific T-cell lines were used to identify T-cell epitopes of Pru p 3. Pru p 3-specific T-cell clones (TCCs) were analyzed for allergen-induced secretion of IL-4, IFN-gamma, and IL-10 and expression of the integrin beta7, a receptor critical for gut homing. RESULTS: No difference in T-cell responses of Italian and Spanish patients was found. Among several T cell-activating regions, Pru p 3(13-27), Pru p 3(34-48), Pru p 3(43-57), and Pru p 3(61-75) were most frequently recognized in 18 Pru p 3-specific T-cell lines. The majority of 32 Pru p 3-specific TCCs belonged to the T(H)2 subset. In contrast to TCCs specific for other plant food and pollen allergens, only a limited number of Pru p 3-specific TCCs produced significant amounts of IL-10. The expression of integrin beta7 on Pru p 3-specific TCCs was comparable with that observed on peanut-specific TCCs and higher compared with that seen in different pollen-specific TCCs. CONCLUSION: The T-cell response to Pru p 3 is dominated by T(H)2 cells presumably primed in the gut. The identification of relevant T cell-activating regions provides a basis for engineering hypoallergenic variants of Pru p 3 with less IgE binding and retained T-cell stimulatory capacity for safe immunotherapy of peach allergy.

Relevance of IgE binding to short peptides for the allergenic activity of food allergens.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Detection of allergen specific immunoglobulins by microarrays coupled to microfluidics.

Allergen microarrays are under development for a component-resolved diagnosis of Type I (IgE-mediated) allergies. Here we report an improved microarray coupled to microfluidics for the detection of allergen specific immunoglobulin E (IgE). The signal intensity for IgE detection in serum has been improved by using glass slides coated with a novel poly[DMA-co-NAS] brush copolymer which is able to immobilize allergens in their native conformation and by carrying out the incubation step in dynamic conditions. The assay, fully automated, was performed in a microcell, using a software-controlled fluidic processor, to bring assay reagents on the surface of the array. Microfluidics turns the binding between serum immunoglobulins and immobilized allergens from a diffusion-limited to a kinetic-limited process by ensuring an efficient mixing of serum samples on the surface of the microarray. As a result of this, the binding of high affinity IgE antibodies is enhanced whereas that of low affinity IgG antibodies, which are present at higher concentration, is impaired paving the way to more accurate and sensitive results.

Hazelnut (Corylus avellana) vicilin Cor a 11: molecular characterization of a glycoprotein and its allergenic activity.

In Europe, hazelnuts (Corylus avellana) are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterized. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterization of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Although MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS showed that one of two potential glycosylation sites of Cor a 11 was glycosylated, CD spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus to analyse the impact of the glycan residues of Cor a 11 on IgE binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitization pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine-release assays were used to determine the allergenic activity of both natural and recombinant Cor a 11 in comparison with Cor a 1, a birch (Betula verrucosa) pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.

Recombinant tropomyosin from Penaeus aztecus (rPen a 1) for measurement of specific immunoglobulin E antibodies relevant in food allergy to crustaceans and other invertebrates.

Immunoglobulin E (IgE)-mediated food allergy to crustaceans and mollusks is relatively common and affected individuals typically react to a range of different species. The only known major allergen of shrimp was first described over 20 years ago and later identified as the muscle protein tropomyosin. This protein may be useful as a defined and relevant diagnostic marker for allergic sensitization to invertebrate foods. In order to generate an assay reagent suitable for this purpose, tropomyosin from the shrimp Penaeus aztecus (Pen a 1) was produced as a recombinant protein in Escherichia coli and characterized with respect to IgE antibody binding properties in comparison to natural shrimp tropomyosin. Hexahistidine-tagged rPen a 1 accumulated as a predominantly soluble protein in the E. coli expression host and a two-step chromatographic procedure provided a high yield of pure and homogeneous protein. rPen a 1 displayed chromatographic and folding characteristics similar to those of purified natural shrimp tropomyosin. Serum preincubation with serial protein dilutions revealed similar capacity of recombinant and natural tropomyosin to compete with immobilized shrimp extract for IgE binding. rPen a 1 was further shown to extensively and specifically compete for IgE binding to extracts of other crustacean species, house dust mite and German cockroach.

A food matrix reduces digestion and absorption of food allergens in vivo.

SCOPE: Food allergy is caused by primary (class 1) food allergens, e.g. Bos d 5 (cow's milk) and Cor a 8 (hazelnut) or secondary (class 2) food allergens, e.g. Mal d 1 (apple). The latter cannot sensitize susceptible individuals but can cause allergy due to immunological cross-reactivity with homologous respiratory allergens. Here, we studied the effects of food matrix on gastrointestinal proteolysis, epithelial transport and in vivo absorption of class 1 and class 2 food allergens. METHODS AND RESULTS: Mal d 1 lost its IgE-reactivity immediately after simulated gastric digestion whereas Bos d 5 and Cor a 8 did not. Only Cor a 8 maintained IgE-binding capacity after simulated intestinal proteolysis. The presence of hazelnut and peanut extracts, which served as protein-rich model food matrices, delayed gastrointestinal degradation and reduced epithelial transport rates of all allergens through CaCo-2 monolayers. Finally, IgE-reactive allergens were assessed at different time points in sera from rats fed with all three allergens with or without hazelnut extract. The levels of all allergens peaked 2 h after animals were fed without matrix and increased over 8 h after feeding. CONCLUSIONS: A protein-rich food matrix delays gastrointestinal digestion and epithelial transport of food allergens and thereby may affect their sensitizing capacity and clinical symptoms.

Wine allergy in a wine-growing district: tolerance induction in a patient with allergy to grape lipid-transfer protein.

BACKGROUND: : An IgE-mediated allergy against a lipid-transfer protein of grapes was the cause of repeated severe anaphylaxis in a patient after consumption of grapes, wine, and raisins. OBJECTIVE: : Although the patient was aware of her grape allergy, avoidance proved difficult and accidental anaphylaxis occurred. Furthermore, wine allergy in a wine-growing district means a non-negligible restriction of quality of life. METHODS: : Although there is little data on specific oral tolerance induction (SOTI) in lipid-transfer protein (LTP) allergy, SOTI with increasing doses starting from approximately 20 mg of grapes was done. For follow-up, skin tests, grape-specific IgE and IgG4, basophil activation tests, and immunoblotting were performed. RESULTS: : Within 3 days the patient reached tolerance to the daily maintenance dose of 20 g of grapes (about 3 grape pieces) without anaphylaxis symptoms. Two months later, a controlled challenge with a total of 66.5 mL of white wine was tolerated. Grape-specific IgE stayed stable at 2.37 kU/L (class 2) and grape-specific IgG4 was first detectable 21 months after SOTI. Prick-to-prick skin tests continued to be positive to grapes, to raisins, and to white and red wine. The basophil activation test still showed strong IgE-mediated activation of basophils after stimulation with grape extract. Immunoblotting still detected IgE binding to a 8-kDa protein. CONCLUSIONS: : We performed SOTI in a patient with severe IgE-mediated allergy against the LTP Vit v 1 of grapes and reduced the risk of anaphylaxis because of accidental intake of any kind of grapes. However, underlying mechanisms of SOTI and maintenance of the established tolerance are still not known.

High level expression, purification and physico- and immunochemical characterisation of recombinant Pen a 1: a major allergen of shrimp.

Well-characterised and immunologically active recombinant allergens are of eminent importance for improvement of diagnostic tools and immunotherapy of allergic diseases. The use of recombinant allergens has several advantages such as the more precise quantification of the active substance compared to allergen extracts and the reduced risk of contamination with other allergenic proteins compared to purified natural allergens. Optimised standard protocols for expression and purification and a detailed physico-chemical characterisation of such recombinant allergens are necessary to ensure consistent quality and comparability of results obtained with recombinant material. In this study the major allergen Pen a 1 of brown shrimp (Penaeus aztecus) was expressed in E. coli and purified in two steps by immobilised metal chelate-affinity chromatography (IMAC) and size-exclusion chromatography. Identity and purity were verified with N-terminal sequencing and peptide mass fingerprinting. Circular dichroism and NMR-spectroscopy indicated an alpha-helical flexible structure of rPen a 1 which is in accordance with the known structure of tropomyosins. Finally, the recombinant allergen proved to be immunologically reactive in IgE Western blot analysis and ELISA. This study provides a protocol for the preparation of recombinant shrimp tropomyosin in standardised quality.

Expression and characterization of three important panallergens from hazelnut.

Several hazelnut allergens with different clinical relevance and crossreactive properties have been identified and characterized so far. The aim of this study was to develop protocols for producing relatively large amounts of three recombinant hazelnut allergens Cor a 1.04, Cor a 2, and Cor a 8 in a folded and immunologically active form. The availability of well-characterized, pure recombinant allergens will improve diagnostic in vitro tests for food allergy, by allowing a highly sensitive component resolved diagnosis. Depending on the individual hazelnut allergen, protocols for heterologous production - either as fusion or nonfusion protein - were developed to obtain homogenous protein batches. The resulting proteins were purified by a two-step FPLC method and their IgE antibody reactivity was verified. Identity was verified by N-terminal sequencing and MALDI-TOF-MS analysis. Their secondary and tertiary structure was controlled by circular dichroism (CD)-spectroscopy and NMR analysis. Decisions on the strategies for expression and purification of allergens on a large scale were made on a case by case basis: Preparation of rCor a 1.04 and rCor a 2 as fusion proteins in E. coli from inclusion bodies resulted in approximately 10 mg pure protein per liter whereas rCor a 8 expression in yeast as nonfusion protein yielded 30 mg/L.

Production and characterization of an allergen panel for component-resolved diagnosis of celery allergy.

In celery a relevant food allergen source, three allergens have been identified so far: Api g 1 and Api g 4, and one glycosylated protein, Api g 5. For component-resolved food allergy diagnosis high amounts of well-defined allergens are needed. Depending on the individual celery allergen, protocols for heterologous production and purification from natural source, respectively, were established to obtain homogenous protein batches. Afterwards the purified recombinant allergens, Api g 1, Api g 4 and natural Api g 5 were characterized regarding their structural integrity and immunological activity. Therefore, several methods were applied. Proteins were identified by partial N-terminal sequencing, protein mass was verified by MS and sequence integrity by MALDI-TOF and N-terminal sequencing after tryptic digestion. Presence of isoforms in natural allergen preparations was identified by 2-DE. Secondary and tertiary structures were evaluated by circular dichroism (CD) spectroscopy and NMR analysis. Finally, IgE binding capacity was verified using selected sera from celery allergic patients in IgE immunoblots and IgE ELISA. These well-defined celery allergens will be used to prove the concept of component-resolved diagnosis and will contribute to improve food allergy diagnosis in the future.

Reduced allergenic potency of VR9-1, a mutant of the major shrimp allergen Pen a 1 (tropomyosin).

The major shrimp allergen, tropomyosin, is an excellent model allergen for studying the influence of mutations within the primary structure on the allergenic potency of an allergen; Pen a 1 allows systematic evaluation and comparison of Ab-binding epitopes, because amino acid sequences of both allergenic and nonallergenic tropomyosins are known. Individually recognized IgE Ab-binding epitopes, amino acid positions, and substitutions critical for IgE Ab binding were identified by combinatorial substitution analysis, and 12 positions deemed critical were mutated in the eight major epitopes. The mutant VR9-1 was characterized with regard to allergenic potency by mediator release assays using sera from shrimp-allergic subjects and sera from BALB/c, C57BL/6J, C3H/HeJ, and CBA/J mice sensitized with shrimp extract using alum, cholera toxin, and Bordetella pertussis, as adjuvants. The secondary structure of VR9-1 was not altered; however, the allergenic potency was reduced by 90-98% measuring allergen-specific mediator release from humanized rat basophilic leukemia (RBL) cells, RBL 30/25. Reduced mediator release of RBL-2H3 cells sensitized with sera from mice that were immunized with shrimp extract indicated that mice produced IgE Abs to Pen a 1 and to the same epitopes as humans did. In conclusion, data obtained by mapping sequential epitopes were used to generate a Pen a 1 mutant with significantly reduced allergenic potency. Epitopes that are relevant for human IgE Ab binding are also major binding sites for murine IgE Abs. These results indicate that the murine model might be used to optimize the Pen a 1 mutant for future therapeutic use.

Biological activity of IgE specific for cross-reactive carbohydrate determinants.

BACKGROUND: The clinical relevance of IgE specific for cross-reactive carbohydrate determinants (CCDs) has been a matter of controversy. Until now, no convincing experiments have been performed to test the biologic significance of individual multivalent allergens that carry multiple carbohydrate epitopes. OBJECTIVE: We sought to contribute to the understanding of the role of CCD-specific IgE antibodies and to study whether CCD-specific IgE antibodies are able to activate basophils using different multivalent glycoproteins as antigens. METHODS: Purified natural tomato beta-fructofuranosidase (nLyc e 2) and rLyc e 2.02 expressed in Escherichia coli were compared by means of histamine release tests. In addition, native and deglycosylated horseradish peroxidase and a neoglycoprotein consisting of a defined glycopeptide (Manalpha1-6[Xylbeta1-2]Manbeta1-4GlcNAcbeta1-4[Fucalpha1-3]GlcNAc) coupled to BSA were used in histamine release assays using stripped basophils from normal donors resensitized with IgE from CCD-reactive patients with food allergy to tomato. RESULTS: Ten CCD-positive and 2 CCD-negative sera from patients with tomato allergy underwent histamine release testing by using the glycoproteins and nonglycosylated controls as antigens, respectively. All sera induced histamine release with tomato extract (up to 100%), confirming the allergic status of the donors. Four of the CCD-positive sera induced releases ranging from 12% to 82% with all of the glycoproteins but not with the nonglycosylated or monovalent controls. All other sera showed no response or only very weak response to the glycoproteins. CONCLUSION: Approximately one third of the CCD-positive sera from patients with tomato allergy have biologically relevant CCD-specific IgE antibodies. Therefore the general claim that CCD-specific IgE is clinically irrelevant has to be reconsidered critically. Hence IgE specific for CCDs should be taken into consideration in the diagnosis and therapy of certain allergies. In the subgroup of patients sensitized to CCDs, the use of natural allergens should be preferred over the use of recombinant allergens expressed in prokaryotic organisms.

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

Strong allergenicity of Pru av 3, the lipid transfer protein from cherry, is related to high stability against thermal processing and digestion.

BACKGROUND: Nonspecific lipid transfer proteins (nsLTPs) have been identified as major fruit allergens in patients from the Mediterranean area. Sensitization to nsLTPs is accompanied by severe reactions, possibly because of specific biophysical and biochemical properties of this allergen family. OBJECTIVE: To assess the protein stability and allergenic potency of nsLTP from fruits in comparison with birch pollen-related allergens from the same allergenic source. METHODS: Stability of natural and recombinant cherry allergens Pru av 3 (nsLTP), Pru av 1 (Bet v 1 homologue), and Pru av 4 (profilin) to pepsin digestion and to thermal processing and stability of allergens in skin prick test reagents was investigated by immunoblotting and/or circular dichroism spectroscopy. Moreover, allergenicity of processed and fresh fruits in regard to Pru av 1 and Pru av 3 was analyzed by histamine release assays. RESULTS: Lipid transfer proteins showed the highest resistance to digestion by pepsin (rPru av 3 > rPru av 1 > rPru av 4). Immunologically active Pru av 3 was detectable after 2 hours of digestion by pepsin, whereas IgE reactivity of Pru av 1 and Pru av 4 was abolished within less than 60 minutes. In contrast with Pru av 1, IgE reactivity to nsLTPs was not diminished in thermally processed fruits, and secondary structures of purified Pru av 3 were more resistant to heating. Moreover, nsLTPs were stable components in skin prick test reagents. Histamine release assays confirmed the strong allergenicity of nsLTPs, which was not affected by protease treatment or thermal processing of fruits. CONCLUSION: In contrast with birch pollen-related allergens, nsLTPs are highly stable to pepsin treatment and thermal processing and show higher allergenic potency. Therefore, nsLTPs have the potential to act as true food allergens, probably eliciting severe systemic reactions by reaching the intestinal mucosa in an intact and fully active form.