Walnut Allergy Across Europe: Distribution of Allergen Sensitization Patterns and Prediction of Severity.

BACKGROUND: Walnut allergy is common across the globe, but data on the involvement of individual walnut components are scarce. OBJECTIVES: To identify geographical differences in walnut component sensitization across Europe, explore cosensitization and cross-reactivity, and assess associations of clinical and serological determinants with severity of walnut allergy. METHODS: As part of the EuroPrevall outpatient surveys in 12 European cities, standardized clinical evaluation was conducted in 531 individuals reporting symptoms to walnut, with sensitization to all known walnut components assessed in 202 subjects. Multivariable Lasso regression was applied to investigate predictors for walnut allergy severity. RESULTS: Birch-pollen-related walnut sensitization (Jug r 5) dominated in Northern and Central Europe and lipid transfer protein sensitization (Jug r 3) in Southern Europe. Profilin sensitization (Jug r 7) was prominent throughout Europe. Sensitization to storage proteins (Jug r 1, 2, 4, and 6) was detected in up to 10% of subjects. The walnut components that showed strong correlations with pollen and other foods differed between centers. The combination of determinants best predicting walnut allergy severity were symptoms upon skin contact with walnut, atopic dermatitis (ever), family history of atopic disease, mugwort pollen allergy, sensitization to cat or dog, positive skin prick test result to walnut, and IgE to Jug r 1, 5, 7, or carbohydrate determinants (area under the curve = 0.81; 95% CI, 0.73-0.89). CONCLUSIONS: Walnut-allergic subjects across Europe show clear geographical differences in walnut component sensitization and cosensitization patterns. A predictive model combining results from component-based serology testing with results from extract-based testing and information on clinical background allows for good discrimination between mild to moderate and severe walnut allergy.

Estimating the Risk of Severe Peanut Allergy Using Clinical Background and IgE Sensitization Profiles.

Background: It is not well-understood why symptom severity varies between patients with peanut allergy (PA). Objective: To gain insight into the clinical profile of subjects with mild-to-moderate and severe PA, and investigate individual and collective predictive accuracy of clinical background and IgE to peanut extract and components for PA severity. Methods: Data on demographics, patient history and sensitization at extract and component level of 393 patients with probable PA (symptoms ≤ 2 h + IgE sensitization) from 12 EuroPrevall centers were analyzed. Univariable and penalized multivariable regression analyses were used to evaluate risk factors and biomarkers for severity. Results: Female sex, age at onset of PA, symptoms elicited by skin contact with peanut, family atopy, atopic dermatitis, house dust mite and latex allergy were independently associated with severe PA; birch pollen allergy with mild-to-moderate PA. The cross-validated AUC of all clinical background determinants combined (0.74) was significantly larger than the AUC of tests for sensitization to extract (0.63) or peanut components (0.54-0.64). Although larger skin prick test wheal size, and higher IgE to peanut extract, Ara h 1 and Ara h 2/6, were associated with severe PA, and higher IgE to Ara h 8 with mild-to-moderate PA, addition of these measurements of sensitization to the clinical background model did not significantly improve the AUC. Conclusions: Models combining clinical characteristics and IgE sensitization patterns can help establish the risk of severe reactions for peanut allergic patients, but clinical background determinants are most valuable for predicting severity of probable PA in an individual patient.

In vivo Induction of Functional Inhibitory IgG Antibodies by a Hypoallergenic Bet v 1 Variant.

Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients' first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (104 to 106) were induced, which cross-reacted with wild-type Bet v 1, and the homologous allergens Mal d 1 and Cor a 1. Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT.

Rational Design, Structure-Activity Relationship, and Immunogenicity of Hypoallergenic Pru p 3 Variants.

SCOPE: Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy. METHODS AND RESULTS: Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild-type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long-term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE-binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross-inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate-based adjuvant formulation in a mouse model. CONCLUSIONS: An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy.

Hypoallergenic molecules for subcutaneous immunotherapy.

Although a large part of the population suffers from allergies, a cure is not yet available. Allergen-specific immunotherapy (AIT) offers promise for these patients. AIT has proven successful in insect and venom allergies; however, for food allergy this is still unclear. In this editorial we focus on the recent advances in a proof of concept study in food allergy, FAST (Food allergy specific immunotherapy), which may increase interest within the biomolecular and pharmaceutical industry to embark on similar projects of immunology driven precision medicine within the allergy field.

Hazelnut allergy across Europe dissected molecularly: A EuroPrevall outpatient clinic survey.

BACKGROUND: Hazelnut allergy is birch pollen-driven in Northern/Western Europe and lipid transfer protein-driven in Spain and Italy. Little is known about other regions and other allergens. OBJECTIVE: Establishing a molecular map of hazelnut allergy across Europe. METHODS: In 12 European cities, subjects reporting reactions to hazelnut (n = 731) were evaluated and sensitization to 24 foods, 12 respiratory allergen sources, and latex was tested by using skin prick test and ImmunoCAP. A subset (124 of 731) underwent a double-blind placebo-controlled food challenge to hazelnut. Sera of 423 of 731 subjects were analyzed for IgE against 7 hazelnut allergens and cross-reactive carbohydrate determinants by ImmunoCAP. RESULTS: Hazelnut allergy was confirmed in 70% of those undergoing double-blind placebo-controlled food challenges. Birch pollen-driven hazelnut sensitization (Cor a 1) dominated in most cities, except in Reykjavik, Sofia, Athens, and Madrid, where reporting of hazelnut allergy was less frequent anyhow. In Athens, IgE against Cor a 8 dominated and strongly correlated with IgE against walnut, peach, and apple and against Chenopodium, plane tree, and mugwort pollen. Sensitization to seed storage proteins was observed in less than 10%, mainly in children, and correlated with IgE to nuts, seeds, and legumes. IgE to Cor a 12, observed in all cities (10% to 25%), correlated with IgE to nuts, seeds, and pollen. CONCLUSIONS: In adulthood, the importance of hazelnut sensitization to storage proteins, oleosin (Cor a 12), and Cor a 8 is diluted by the increased role of birch pollen cross-reactivity with Cor a 1. Cor a 8 sensitization in the Mediterranean is probably driven by diet in combination with pollen exposure. Hazelnut oleosin sensitization is prevalent across Europe; however, the clinical relevance remains to be established.

Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy.

BACKGROUND: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. OBJECTIVES: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. METHODS: Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. RESULTS: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. CONCLUSION: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.

Modified allergens and their potential to treat allergic disease.

Allergen-specific immunotherapy is the only treatment of allergic diseases that aims at modifying the underlying immune mechanism. Current protocols are long and at risk of anaphylactic reactions. The main aim of current research is decreasing the risk of side effects and increasing efficacy, in particular targeting reduction of treatment duration. Since the advent of molecular biology, extracts can be replaced by recombinant hypo-allergens, peptides, or fusion proteins. In addition, different routes of administration are being pursued as well as the addition of new adjuvants that are targeted at skewing the immune system away from a Th2 to a more Th1 or regulatory T cell phenotype. In this review, we summarize the recent advances in this field focusing on the allergen modifications and new adjuvants.

Oil body-associated hazelnut allergens including oleosins are underrepresented in diagnostic extracts but associated with severe symptoms.

BACKGROUND: Oil body-associated allergens such as oleosins have been reported for important allergenic foods such as peanut, sesame and hazelnut. Here we investigate whether oil body associated proteins (OAPs) are linked with specific clinical phenotypes and whether they are represented in skin prick test (SPT) reagents. METHODS: A hazelnut OAP fraction was characterized by mass-spectrometry (MS) to identify its major constituents. Polyclonal rabbit antibodies were generated against hazelnut OAPs. The presence of OAPs in commercially available hazelnut SPTs was studied by immunoblot and spiking experiments. OAP-specific IgE antibodies were measured in sera from patients with a convincing history of hazelnut allergy by RAST (n = 91), immunoblot (n = 22) and basophil histamine release (BHR; n = 14). RESULTS: Hazelnut OAPs were analysed by MS and found to be dominated by oleosins at ~14 and ~17 kDa, and a 27 kDa band containing oleosin dimers and unidentified protein. In 36/91 sera specific IgE against hazelnut OAPs was detected, and confirmed to be biologically active by BHR (n = 14). The majority (21/22) recognized the oleosin bands at 17 kDa on immunoblot, of which 11 exclusively. These OAP-specific IgE responses dominated by oleosin were associated with systemic reactions to hazelnut (OR 4.24; p = 0.015) and negative SPT (χ2 6.3, p = 0.012). Immunoblot analysis using OAP-specific rabbit antiserum demonstrated that commercial SPT reagents are virtually devoid of OAPs, sometimes (3/9) resulting in false-negative SPT. Spiking of SPT reagents with OAP restored serum IgE binding of these false-negative patients on immunoblot at mainly 17 kDa. CONCLUSION: Hazelnut allergens found in oil bodies dominated by oleosin are associated with more severe systemic reactions and negative SPT. Defatted diagnostic extracts are virtually devoid of these allergens, resulting in poor sensitivity for detection of IgE antibodies against these clinically relevant molecules.

Oral delivery of a probiotic induced changes at the nasal mucosa of seasonal allergic rhinitis subjects after local allergen challenge: a randomised clinical trial.

OBJECTIVE: To determine effects of probiotic consumption on clinical and immunological parameters of seasonal allergic rhinitis (SAR) in an out-of-season single nasal allergen challenge. METHODS: In a study registered at ClinicalTrials.Gov (NCT01123252), a 16-week dietary intervention was undertaken in 60 patients with allergic rhinitis (>16 years old). Using a double-blinded, placebo-controlled anonymised design, the patients were divided equally into two groups. One group was given a dairy drink containing Lactobacillus casei Shirota to ingest daily while the other consumed a similar drink without bacteria. Participants attended the clinic on two consecutive days before the intervention and then again at the end of the study period. On the first day of each 2-day visit, following clinical examination, assessments were made of total nasal symptoms scores and peak nasal inspiratory flow. Nasal scrapings, nasal lavage and blood were collected for laboratory analyses of cellular phenotypes, soluble mediator release and in vitro responses to pollen allergen. These procedures were repeated 24 hours following nasal allergen challenge. RESULTS: Prior to and following intervention there were no detectable differences between study groups in measured clinical outcome. After intervention, there were differences between groups in their percentages of CD86+ epithelial cells (p = 0.0148), CD86+CD252+ non-epithelial cells (p = 0.0347), sIL-1RII release (p = 0.0289) and IL-1ß (p = 0.0224) levels at the nasal mucosa. Delivery of probiotic also suppressed production of sCD23 (p = 0.0081), TGF-ß (p = 0.0283) and induced increased production of IFN-γ (p = 0.0351) in supernatants of cultured peripheral blood. CONCLUSIONS & CLINICAL RELEVANCE: This study did not show significant probiotic-associated changes with respect to the primary clinical endpoint. An absence of overt clinical benefit may be due to an inability of single nasal challenges to accurately represent natural allergen exposure. Nevertheless, oral delivery of probiotics produced changes of the immunological microenvironment at the nasal mucosa in individuals affected by SAR. TRIAL REGISTRATION: ClinicalTrials.Gov NCT01123252.

Sensitization to Cor a 9 and Cor a 14 is highly specific for a hazelnut allergy with objective symptoms in Dutch children and adults.

BACKGROUND: Component-resolved diagnosis has been shown to improve the diagnosis of food allergy. OBJECTIVE: We sought to evaluate whether component-resolved diagnosis might help to identify patients at risk of objective allergic reactions to hazelnut. METHOD: A total of 161 hazelnut-sensitized patients were included: 40 children and 15 adults with objective symptoms on double-blind, placebo-controlled food challenges (DBPCFCs) and 24 adults with a convincing objective history were compared with 41 children and 41 adults with no or subjective symptoms on DBPCFCs (grouped together). IgE levels to hazelnut extract and single components were analyzed with ImmunoCAP. RESULTS: IgE levels to hazelnut extract were significantly higher in children with objective than with no or subjective symptoms. In 13% of children and 49% of adults with hazelnut allergy with objective symptoms, only sensitization to rCor a 1.04 was observed and not to other water-soluble allergens. Sensitization to rCor a 8 was rare, which is in contrast to rCor a 1. Sensitization to nCor a 9, rCor a 14, or both was strongly associated with hazelnut allergy with objective symptoms. By using adapted cutoff levels, a diagnostic discrimination between severity groups was obtained. IgE levels to either nCor a 9 of 1 kUA/L or greater or rCor a 14 of 5 kUA/L or greater (children) and IgE levels to either nCor a 9 of 1 kUA/L or greater or rCor a 14 of 1 kUA/L or greater (adults) had a specificity of greater than 90% and accounted for 83% of children and 44% of adults with hazelnut allergy with objective symptoms. CONCLUSION: Sensitization to Cor a 9 and Cor a 14 is highly specific for patients with objective symptoms in DBPCFCs as a marker for a more severe hazelnut allergic phenotype.

FAST: towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies.

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity.

SCOPE: Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1. METHODS AND RESULTS: Ara h 1 was purified from either raw (N-Ara h 1) or roasted (R-Ara h 1) peanuts. Boiling (100°C 15 min; H-Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod-like branched aggregates with reduced IgE-binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G-Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, H- and G-Ara h1 retained the T-cell reactivity of N-Ara h 1. R-Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE-binding capacity of the native protein. CONCLUSION: Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts.

Effect of heating and glycation on the allergenicity of 2S albumins (Ara h 2/6) from peanut.

BACKGROUND: Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut. METHODOLOGY/PRINCIPAL FINDINGS: Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6. CONCLUSIONS/SIGNIFICANCE: Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.

Sensitization prevalence, antibody cross-reactivity and immunogenic peptide profile of Api g 2, the non-specific lipid transfer protein 1 of celery.

BACKGROUND: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. METHODOLOGY: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. RESULTS: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. CONCLUSIONS: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.