Short or common ragweed (Ambrosia artemisiifolia), belonging to the plant family of Asteraceae, is an annual weed with pollination peak levels in late summer. Epidemiological studies revealed a sensitization prevalence of 23%–32.8% for the US population, whereas in European countries the prevalence shows more variety, for example, 3.5% in Italy and 54% in Hungary.1 Due to the rapid dispersal of ragweed, its massive pollen production, and high allergenic potential, ragweed allergy is developing into a significant global health concern. To date, 11 allergens have been recorded in the official IUIS allergen database, with Amb a 1 representing the most clinically relevant allergen with sensitization rates >90%.2, 3 At present, allergen-specific immunotherapy (AIT) represents the only treatment for respiratory allergies leading to long-lasting clinical benefits up to permanent immune tolerance after treatment discontinuation. Subcutaneous immunotherapy (SCIT) generally shows more extensive effects compared with sublingual immunotherapy (SLIT)4; however, at present no commercially SCIT is available for ragweed. Therefore, a subcutaneous chemically modified ragweed-based immunotherapy product (MRE) is being developed. In the present study, the immunogenicity and allergenicity of MRE in comparison with a common ragweed extract (RE) were evaluated. Modified ragweed-based immunotherapy product was generated by cross-linking of standardized ragweed pollen extract with glutaraldehyde followed by adsorption to aluminum hydroxide (Al(OH)3). Mass spectrometry (MS) analyses of MRE verified the conservation of all major allergens and minor allergens with the exception of Amb a 9 and Amb a 10. Furthermore, various peptides of the five known Amb a 1 isoforms could be clearly identified (Table S1). Dynamic light scattering (DLS) analyses showed that MRE has a hydrodynamic radius of 10.78 nm. This radius and the type of peak formation indicate a single aggregated population, verifying that chemical modification was successful (Figure S1A).5, 6 Allergenicity of MRE vs. RE was assessed by mediator release experiments using ragweed-allergic patient sera. Sera were obtained from 15 subjects (8 males and 7 females, mean age: 35.5) with clinical history of ragweed pollen allergy, positive skin prick tests, and comparable total IgE ImmunoCAP values. Immunoblot analysis using RE confirmed IgE reactivity toward ragweed allergens (Figure S2). Experiments using anonymized serum samples of ragweed-allergic patients from Austria were approved by the Ethics Committee of the Medical University of Vienna (No. 712/2010), and informed written consents were obtained. Subjects' clinical and serologic characteristics are summarized in Table S2. Mediator release assays were performed using RBL-2H3 cells carrying the human FcεRI alpha chain.7 RBL-2H3 cells were passively sensitized with human sera. Serial dilutions of RE or MRE were used to trigger IgE receptor cross-linking followed by allergen-dependent ß-hexosaminidase release from cells into the supernatant. Results revealed that RE induces higher overall mediator release than MRE in ragweed-allergic patients. For patients 14 and 15 (both Amb a 1 non-responders; Figure S2), very limited activation with MRE was observed (Figure S3). Calculations of the antigen concentration at which 25% ß-hexosaminidase release was reached showed that statistically significantly more MRE (mean of 696.96 ng/ml) was needed to induce the same amount of mediator release as RE (mean of 1.35 ng/ml) (Figure 1A). Additionally, IgE-binding capacities of RE and MRE were tested by indirect ELISA using 15 ragweed-allergic patients' sera showing a statistically significant reduction in IgE binding against MRE (Figure 1B). These results suggest a significantly lower allergenic potential of MRE compared with RE, which was further verified by inhibition ELISA using concentration series of RE or MRE, respectively, to inhibit IgE binding to RE (Figure 1C). Immunogenic properties of MRE were evaluated in an in vivo mouse model by measuring induction of RE- or Amb a 1–specific IgG1, IgG2a, IgE, and total IgE levels. Animal experiments were performed according to the guidelines of the Austrian Federal Ministry of Science, Research, and Economy (BMWF-66.012/0017-WF/V/3b/2017). After 4 immunizations with formulated RE and MRE, RE-specific and natural Amb a 1–specific IgG1 levels (Figure 2A,B) were increased for MRE compared with RE. IgG2a was only slightly induced in all treated groups with no significant difference (data not shown). Moreover, total and specific IgE levels (Figure S5A,B,C) were lower in MRE-immunized mice than RE-immunized mice, suggesting a lower risk for allergic side effects during SCIT. These results indicate that MRE not only induces less total and specific IgE compared with RE but is also a stronger inducer of IgG1 antibodies, which are cross-reactive with unmodified ragweed pollen proteins. The allergenic potency of IgE induced by MRE immunizations in comparison with RE immunizations was addressed by murine mediator release experiments. Results showed that immunizations with RE induced slightly higher ß-hexosaminidase release in comparison with MRE-immunized mice at the representative RE concentration of 100 ng/ml (Figure 2F), indicating similar allergenic potency. IgE-facilitated allergen binding (FAB) assays were used to determine the capacity of IgG induced upon immunization of mice to prevent IgE-RE complex formation and revealed that IgG induced upon MRE immunization possesses a higher inhibition capacity than IgG induced by RE immunization (Figure S1B). ELISpot experiments were performed using splenocytes of RE- or MRE-immunized mice collected 1 week after final immunization and restimulated with either RE, MRE, or tissue culture medium to identify induction of inflammatory cytokines. RE and MRE restimulation of splenocytes from mice immunized with either RE or MRE did not induce IL-10–producing cells but significantly increased IFN-γ–secreting cells (in case of RE) (Figure S4A); additionally, IL-4 (Figure S4B)- and IL-5 (Figure S4C)–secreting cells were elevated compared to restimulation with medium. Thereby, RE was a significantly stronger stimulus for the induction of all three cytokines, compared to stimulation with MRE, independent of the immunization regime. Stimulation of splenocytes from mice immunized with sham did not lead to any induction of cytokine-producing cells. In conclusion, it was demonstrated that chemical modification of ragweed significantly reduced IgE binding and mediator release from human sera about 500-fold, suggesting a significantly decreased allergenicity. Furthermore in mouse models, MRE has proved to be a strong inducer for antibodies, which are cross-reactive with unmodified ragweed pollen proteins. Immunization with MRE induced slightly less total and allergen-specific IgE but at the same time induced higher IgG1 levels. Further, in ELISpot experiments MRE induced significantly less IFN-γ and IL-4 and IL-5, in comparison with RE. Taken together, these results demonstrate that MRE is a highly valuable candidate to be further tested as vaccine for the treatment of ragweed pollen allergies. In future studies using an immunotherapy model, the therapeutic efficacy of MRE will be addressed in detail. The authors would like to acknowledge Prof. Barbara Bohle, Medical University of Vienna, for providing patients’ sera. Hanneke P.M van der Kleij is an employee of HAL Allergy. Fatima Ferreira is a member of Scientific Advisory Boards (HAL Allergy, NL; SIAF, Davos, CH; AllergenOnline, USA) and has been supported by the Austrian Science Funds (FWF). Martin Wolf, Michael Hauser, Sara Huber, Sabrina Wildner, Claudia Asam, Heidi Hofer, Peter Briza, and Michael Wallner have no conflict of interest to declare. This study was supported by HAL Allergy BV, The Netherlands. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Allergen‐specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens.

Background Tropomyosins are highly conserved proteins, an attribute that forms the molecular basis for their IgE antibody cross-reactivity. Despite structural similarities, their allergenicity varies greatly between ingested and inhaled invertebrate sources. In this study, we investigated the relationship between the structural stability of different tropomyosins, their endolysosomal degradation patterns and T-cell reactivity. Methods We investigated the differences between four tropomyosins - the major shrimp allergen Pen m 1 and the minor allergens Der p 10 (dust mite), Bla g 7 (cockroach) and Ani s 3 (fish parasite) - in terms of IgE binding, structural stability, endolysosomal degradation and subsequent peptide generation, and T-cell cross-reactivity in a BALB/c murine model. Results Despite their conserved primary structure and consequent IgE co-reactivity, the invertebrate tropomyosins displayed different protein stabilities. Pen m 1 and Ani s 3, but not Der p 10 and Bla g 7 elicited differential melting temperatures that were pH-dependent. Endolysosomal experiments demonstrated differential degradation, as a function of stability, generating different peptide repertoires. Pen m 1 T-cell clones, with specificity for sequences highly conserved in all four tropomyosins, did not proliferate with Der p 10, Bla g 7 and Ani s 3, indicating that these peptides were not naturally produced for other invertebrate tropomyosins. Conclusions Our data suggest that, although invertebrate tropomyosins exhibit a high degree of IgE cross-reactivity due to conserved B-cell epitopes, they do not necessarily share identical cross-reactive T-cell epitopes. This is likely due to differential endolysosomal processing as a function of different structural stabilities.

in infrequent phenotypes such as isolated angioedema by SRs and multiple SRs, which may be misdiagnosed as CRs. This procedure is useful not only for reducing the number of patients who are unnecessarily avoiding NSAIDs but also for extending their therapeutic possibilities. Although such accurate diagnosis requires facilities and trained personal in this field, it will improve the allergological workup and, consequently, patients' management.

More than twenty different isoforms of Bet v 1, the major birch pollen allergen, have been identified, sharing an amino acid sequence identity of 95% and an almost identical tertiary structure.1 Despite their structural similarities, the isoforms display remarkable different immunogenic properties and IgE-binding capacities.2 Bet v 1 isoforms were recently shown to differ in ligand binding concerning small hydrophobic plant mediators, which could relate to the diverging immunogenic and allergenic properties of the Bet v 1 isoforms.3 We found that Bet v 1a (Bet v 1.0101), structurally comparable to human lipocalin-2, is able to bind iron via catechol-based siderophores in its internal cavity.4 When incubated with human immune cells, only the unloaded apo-Bet v 1 molecule caused Th2 cells to secrete IL-13.4 Our search for other ligands able to induce immunomodulation, supported by data from literature and in silico docking calculations, led us to the major vitamin A metabolite retinoic acid (RA). RA has not only intrinsic immunomodulatory properties,5 it is also able to abrogate the Th2 immunogenicity of the major milk allergen Bos d 5 when in holo-form.6 In the present study, we hence concentrated on the ability of Bet v 1a and of the hypoallergenic isoform Bet v 1d (Bet v1.0102) to bind RA in their internal cavity and the subsequent effects on their allergenic potential. First, we investigated whether RA at all is able to bind into the cavity of Bet v 1 utilizing in silico docking analysis and an in vitro ANS competition assay (Figure 1A, D). In silico calculations, using the crystal structure of the Bet v 1-naringenin complex (PBD entry 4A87) for Bet v 1a (Figure 1A) and a homology model template based on PDB entry 4MNS for Bet v 1d (Figure 1D), revealed an identical affinity energy of −8.7 kcal/mol for both isoforms, corresponding to a dissociation constant of 0.364 µmol/L. The close-up view of the RA-binding site in the intramolecular cavity of Bet v 1a (Figure 1A) and Bet v 1d (Figure 1D) shows a hydrogen bond between oxygen atoms of RA and Asp27. The second hydrogen bond relates to residue Tyr81 and residue Lys54 for Bet v 1a and Bet v 1d, respectively. Our in silico findings were corroborated by an in vitro ANS competition assay showing that RA dose-dependently displaced ANS from both Bet v 1a (Figure 1 A) as well as Bet v 1d (Figure 1D), which indicates that principally both Bet v 1 isoforms are able to bind RA in their hydrophobic cavity. Next, we investigated whether RA binding has an influence on the IgE-binding capacity of the Bet v 1 isoforms. In ELISA holo-Bet v 1a (+ ligand RA) showed significantly reduced binding of serum IgE compared to the unloaded apo-Bet v 1a (Figure 1B). Not only the IgE binding but also the IgE cross-linking abilities of Bet v 1a were affected by RA, as holo-Bet v 1a (+ ligand RA) induced significantly less ß-hexosaminidase mediator release from RBL-SX38 cells (Figure 1B). Similarly, mediator release in primary human mast cells, presensitized with IgE from sera of BP-allergic donors, was significantly reduced by holo-Bet v 1a treatment (Figure 1B). In contrast, both apo- and holo-Bet v 1d displayed lower IgE-binding capacity (Figure 1E), and we found no significant differences in mediator release independent whether the unloaded apo- or the RA-loaded holo-form of Bet v 1d was used in the RBL assay or in primary human mast cells (Figure 1E). Subsequent in silico analysis revealed that the IgE-specific effects may be due to epitope masking by ligand RA, as RA binding could interfere with two described IgE-binding B-cell epitope regions in the Bet v 1 structure (Figure S1A). Preliminary in vitro data showed that this effect reached significance with RA only, and not with control ligands epinephrine and catechol (Figure S2). In this context, we also found that protein residues Glu142 and Leu144, representing important T-cell epitope residues of Bet v 1a within the major T-cell epitope, are in favourable position to interact with RA (Figure S1B). Thus, we hypothesized that also cellular immune responses could be influenced by ligand RA binding. Therefore, we incubated PBMCs from BP-allergic donors with unloaded or RA-loaded Bet v 1a and measured the cytokines released into supernatants. PBMCs from BP-allergic donors produced significantly less IFN-γ, IL-13 and IL-10 when stimulated with RA-loaded hyperallergen, holo-Bet v 1a, than with unloaded apo-Bet v 1a (Figure 1C). Even if this was not due to altered endolysosomal stability of Bet v 1a due to RA binding (Figure S3), it seemed so far like RA-loading transforms the hyperallergen Bet v 1a to a hypoallergen with improved tolerogenic capacity of potential implications for allergen immunotherapy. We aimed to challenge this hypothesis in a therapeutic mouse model of birch pollen allergy (Figure S4). Mice were first made allergic against the major birch pollen allergen Bet v 1a and subsequently treated intranasally with either the apo-Bet v 1a, or with the RA-loaded holo-Bet v 1a, or as a control with RA alone. The therapeutic application of holo-Bet v 1a significantly prevented body temperature drop and anaphylactic symptoms upon a specific allergen challenge compared to control mice (Figure 2A). Mice treated with apo-Bet v 1a displayed anaphylactic symptom levels comparable to the control group (Figure 2A). This effect was recorded and visualized in the noninvasive anaphylaxis imaging cage,7 where more constant body temperature and unchanged physical activity were evident in the holo-Bet v 1a treated group (Figure 2B). The alleviated allergic symptoms in holo-Bet v 1a treated mice were accompanied by significantly enhanced allergen-specific IgG2a, IgG2b and IgA serum levels, while apo-Bet v 1a treated mice showed no significant changes compared to control mice (Figure 2C). Allergen-specific IgE levels were not affected by any treatment (Figure 2C). Similar alterations of allergen-specific IgE and IgG responses can be seen during human allergen-specific immunotherapy.8 Systemic immune response was analysed by cytokine expression in supernatants of splenocytes in vitro stimulated with Bet v 1a (Figure 2D). Significant changes were found in spleen cells from holo-Bet v 1a treated mice, showing significantly enhanced IL-10 levels compared to medium control values (Figure 2D). Th1 (IFN-γ) and Th2 (IL-13) responses (Figure 2D) as well as the percentage of CD4 + CD25+ splenic T cells (Figure S5) exhibited no significant changes after apo- or holo-Bet v 1a treatment. Thus, we were able to demonstrate for the first time that loading of RA into the hydrophobic pocket of Bet v 1a reduces its IgE-binding and cross-linking abilities to human primary mast cells in vitro, and alleviates allergic symptoms in vivo. Carotenoids and fatty acids naturally occur in pollen and exhibit anti-oxidant properties.9 The high binding affinity in the nanomolar range of RA into the pocket of Bet v 1 suggests that such molecules might naturally synergize with the birch pollen allergen. When during environmental stress the pathogenesis-related Bet v 1 molecule gets overexpressed, a resulting predominance of insufficiently loaded apo-allergens may contribute to the allergy epidemic. While the underlying mechanisms of immunomodulation by RA-loading are still not fully understood, we propose that ligand binding can be decisive for the development of tolerance and as a future perspective could be helpful to improve immunotherapeutic approaches in birch pollen allergy. We thank Rodolfo Bianchini for scientific exchange and technical advice. Dr Hufnagl, Dr Afify, Nina Braun M.Sc., Dr Hauser, Dr Wiederstein, Dr Wildner, Dr Redegeld, B. Blokhuis, G. Hofstetter, Dr Pali-Schöll and S. Wagner M. Sc. have nothing to disclose. Dr Wallner reports a FWF grant P 23417. Dr Gadermaier reports personal fees from Bencard and personal fees from Compare database, outside the submitted work. Dr Roth-Walter reports grants from Bencard grant award 2018 and personal fees as a research consultant for Biomedical International. R + D GmbH, outside the submitted work; in addition, Dr Roth-Walter has a patent EP2894478, owned by Biomedical Int. R + D issued. Dr Pacios has a patent EP2894478 issued and a patent US15111162 issued. Dr Jensen-Jarolim reports other from Biomedical International R + D GmbH, Austria, grants from Bencard Allergie GmbH, Germany, during the conduct of the study; in addition, Dr Jensen-Jarolim, Dr Roth-Walter and Dr Pacios have a patent EP 2894478 A1, US20160334418 and WO2015104270A1 issued. This work was supported by the SFB F4606-B28 grant of the Austrian Science Fund FWF. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Fag s 1 is a member of the Pathogen Related protein family 10 (PR-10) and can elicit cross-reaction with IgE antibodies produced against the birch pollen allergen Bet v 1. The Nuclear Magnetic Resonance (NMR) structure of Fag s 1 is presented along with its dynamic properties. It shares 66% identity with Bet v 1 and exhibits the expected three α-helices and seven β-sheets arranged as a semi-beta barrel and exposing the residues mapped as the Bet v 1 IgE epitope. The structural dynamics of Fag s 1 were monitored on the fast and intermediate timescales, using relaxation rates. The complex dynamics of Fag s 1 are closely related to the internal cavity, and they modulate IgE and ligand binding.

BackgroundThe clinical benefit of allergen-specific immunotherapy (AIT) involves induction of blocking antibodies. It is not clear if these antibodies function via steric hindrance alone or a combination of levels, avidities, and epitope specificities, and clinical outcome cannot be predicted. We aim to in-depth characterize serum antibody profiles during birch pollen AIT, investigate therapy-induced antibodies for their capacity to block IgE binding to Bet v 1 and correlate data with clinical outcomes.MethodsImmune responses of five birch pollen allergic patients were monitored during the first year of AIT by nasal provocation tests (NPTs), ImmunoCAP, immunoblots, direct and avidity enzyme-linked immunosorbent assays, mediator release assays, facilitated antigen binding (FAB) assays, and inhibition mediator release assays.ResultsThere was no correlation between NPT results and therapy-induced changes in levels (IgE, IgG, IgA, IgM), avidities, or mediator release potency of Bet v 1-specific antibodies. In FAB assays, blocking antibodies initiated upon AIT were shown to prevent formation of Bet v 1-IgE complexes of an indicator serum pool and significantly correlated with clinical readout. Inhibition mediator release assays using patient-specific IgE for passive sensitization revealed therapy-induced blocking capacities with very good correlation to NPT results. Notably, this assay was the only one to detect a non-responder during treatment in this pilot study.ConclusionsClinical outcome of AIT depends on induction of blocking antibodies able to prevent the patient's own IgE from allergen binding. Monitoring of clinical efficacy seems to be best achieved using the inhibition mediator release assay, as development of relevant blocking antibodies can be verified in a patient-tailored manner.

Background: Enhancing the quality and yield of protein production in heterologous expression systems is an important issue for developing new biopharmaceuticals. It has been shown that the dynamics of protein folding is influenced by codon frequencies. As codon usage frequencies are species specific, this can affect heterologous protein expression. In this respect, “codon harmonization,” that is, the usage of synonymous codons with usage frequencies in the host resembling the usage frequencies in the native organism, is a promising strategy. As recombinant proteins are important tools in the area of allergy research, we investigated in this study the influence of codon harmonization on the production of the major birch pollen allergen Bet v 1.0101. Methods: To accomplish this task, parallel production of several batches of rBet v 1, BWT, together with a harmonized variant, BH, was applied. The expression yield of soluble and insoluble protein was assayed via densitometric analysis of ­SDS-PAGEs for every batch. The quality of purified proteins was assessed with a variety of physicochemical methods including mass spectrometry, circular dichroism, dynamic light scattering, Fourier transform infrared spectroscopy, in vitro degradation, and 1-anilino-8-naphthalene sulfonate-binding assays. Patients’ IgE reactivity was tested in enzyme-linked immunosorbent assays and rat basophil mediator release experiments. Results: No significant differences in the ligand-binding capacity and secondary structure elements, as well as, in immunological assays could be found; however, the production yield was drastically increased for BH. Conclusion: We could show that codon harmonization is a powerful method to enhance protein yields in heterologous expression systems and should be considered especially for difficult-to-express proteins.

Nanotechnology is a fast growing field and already a multi-billion dollar market with numerous consumer products.

Allergic diseases are considered a major problem for healthcare systems in both developed and developing countries. House dust mites are well-known triggers of allergic manifestations. While the Dermatophagoides genus is widely distributed globally, Blomia tropicalis is the most prominent mite species in the tropical and subtropical regions of the world. Over the last decades, an increase in sensitization rates to B. tropicalis has been reported, leading to increased research efforts on Blomia allergens. In fact, 8 new allergens have been identified and characterized to different degrees. Here, we provide an overview of recent developments concerning the identification and production of recombinant Blomia allergens, as well as their structural and immunological characterization. Although considerable progress has been achieved, detailed molecule-based studies are still needed to better define the clinical relevance of Blomia allergens. Thus, the establishment of a well-standardized and fully characterized panel of allergens remains a challenge for the development of better diagnosis and therapy of allergic diseases induced by B. tropicalis.

In Northern America and Europe a great number of people are suffering from birch pollen allergy and pollen related food allergies. The trigger for these immunological reactions is the 17.5 kDa major birch pollen allergen Bet v 1, which belongs to the family of PR-10 (pathogenesis-related) proteins. In nature, Bet v 1 occurs as a mixture of various isoforms that possess different immunological properties despite their high sequence identities. Bet v 1.0102 (Bet v 1d), which is investigated here, is a hypoallergenic isoform of Bet v 1 and a potential candidate for allergen-specific immunotherapy. We assigned the backbone and side chain 1H, 13C and 15N resonances of this protein and predicted its secondary structure. The NMR-chemical shift data indicate that Bet v 1.0102 is composed of three α-helices and a seven stranded β-sheet, in agreement with the known structure of the hyperallergenic isoform Bet v 1.0101 (Bet v 1a). Our resonance assignments create the foundation for detailed characterization of the dynamic properties of Bet v 1 isoforms by NMR relaxation measurements.

Background: Allergy vaccines should be easily applicable, safe, and efficacious. For Bet v 1-mediated birch pollen and associated food allergies, a single wild-type allergen does not provide a complete solution.Objective: We aimed to combine immunologically relevant epitopes of Bet v 1 and the 2 clinically most important related food allergens from apple and hazelnut to a single hybrid protein, termed MBC4.Methods: After identification of T cell epitope-containing parts on each of the 3 parental allergens, the hybrid molecule was designed to cover relevant epitopes and evaluated in silico. Thereby a mutation was introduced into the hybrid sequence, which should alter the secondary structure without compromising the immunogenic properties of the molecule.Results: MBC4 and the parental allergens were purified to homogeneity. Analyses of secondary structure elements revealed substantial changes rendering the hybrid de facto nonreactive with patients' serum IgE. Nevertheless, the protein was monomeric in solution. MBC4 was able to activate T-cell lines from donors with birch pollen allergy and from mice immunized with the parental allergens. Moreover, on immunization of mice and rabbits, MBC4 induced cross-reactive IgG antibodies, which were able to block the binding of human serum IgE.Conclusion: Directed epitope rearrangements combined with a knowledge-based structural modification resulted in a protein unable to bind IgE from allergic patients. Still, properties to activate specific T cells or induce blocking antibodies were conserved. This suggests that MBC4 is a suitable vaccine candidate for the simultaneous treatment of Bet v 1 and associated food allergies.

IgE mediated food allergies affect approximately 3-8" of children and 1-3" of the adult population in industrialized countries. Allergic reactions to food can cause serious, sometimes life-threatening reactions, thus food allergies require special medical attention, including accurate diagnostic as well as therapeutic strategies. Physicochemical data on recombinantly produced allergens is important to understand their properties and immunological behavior and to warrant their quality. Presently, there is a huge variety of well-established techniques available for such analyses. For a comprehensive characterization of recombinant allergens, apart from a detailed physicochemical analysis, the immunological properties of these proteins have to be thoroughly investigated. Such data should confirm that the recombinant molecules exhibit identical immunological behavior as their natural counterparts, allowing their use in either diagnosis or treatment. Double-blind placebo-controlled food challenge is considered the gold standard for diagnosis of food allergies. Peanut is considered a common trigger of food-induced anaphylactic reactions and sensitization rates range from 0.5-7.2" among European adults.