Effect of hot air and infrared roasting on hazelnut allergenicity.
Roasting is known to affect the protein profile and allergenicity of hazelnuts (Corylus avellana cv TGL). The aim of the study was to investigate whether roasting techniques based on different heat transfer methods (hot air and infrared), differently affect the protein solubility and the IgE-binding capacities of both the soluble and insoluble hazelnut protein fractions. The immune-reactivity of the Cor a 9, Cor a 11 and Cor a 14 allergens resulted to be stable after roasting at 140 °C, for both types of treatment, while roasting at 170 °C caused a reduction in IgE-binding, which was particularly noticeable after infrared processing, that led to an almost complete disappearance of allergenicity. Microscopical analyses showed that roasting caused cytoplasmic network disruption, with a loss of lipid compartmentalization, as well as an alteration of the structure of the protein bodies and of the cell wall organization.
Crosslinked Recombinant-Ara h 1 Catalyzed by Microbial Transglutaminase: Preparation, Structural Characterization and Allergic Assessment.
As the one of the major allergens in peanut, the allergenicity of Ara h 1 is influenced by its intrinsic structure, which can be modified by different processing. However, molecular information in this modification has not been clarified to date. Here, we detected the influence of microbial transglutaminase (MTG) catalyzed cross-linking on the recombinant peanut protein Ara h 1 (rAra h 1). Electrophoresis and spectroscopic methods were used to analysis the structural changes. The immunoreactivity alterations were characterized by enzyme linked immunosorbent assay (ELISA), immunoblotting and degranulation test. Structural features of cross-linked rAra h 1 varied at different reaction stages. Hydrogen bonds and disulfide bonds were the main molecular forces in polymers induced by heating and reducing. In MTG-catalyzed cross-linking, ε-(γ-glutamyl) lysine isopeptide bonds were formed, thus inducing a relatively stable structure in polymers. MTG catalyzed cross-linking could modestly but significantly reduce the immunoreactivity of rAra h 1. Decreased content of conserved secondary structures led to a loss of protection of linear epitopes. Besides, the reduced surface hydrophobic index and increased steric hindrance of rAra h 1 made it more difficult to bind with antibodies, thus hindering the subsequent allergic reaction.
Microbial signature in IgE-mediated food allergies.
Background: Multiple studies suggest a key role for gut microbiota in IgE-mediated food allergy (FA) development, but to date, none has studied it in the persistent state. Methods: To characterize the gut microbiota composition and short-chain fatty acid (SCFAs) profiles associated with major food allergy groups, we recruited 233 patients with FA including milk (N = 66), sesame (N = 38), peanut (N = 71), and tree nuts (N = 58), and non-allergic controls (N = 58). DNA was isolated from fecal samples, and 16S rRNA gene sequences were analyzed. SCFAs in stool were analyzed from patients with a single allergy (N = 84) and controls (N = 31). Results: The gut microbiota composition of allergic patients was significantly different compared to age-matched controls both in α-diversity and β-diversity. Distinct microbial signatures were noted for FA to different foods. Prevotella copri (P. copri) was the most overrepresented species in non-allergic controls. SCFAs levels were significantly higher in the non-allergic compared to the FA groups, whereas P. copri significantly correlated with all three SCFAs. We used these microbial differences to distinguish between FA patients and non-allergic healthy controls with an area under the curve of 0.90, and for the classification of FA patients according to their FA types using a supervised learning algorithm. Bacteroides and P. copri were identified as taxa potentially contributing to KEGG acetate-related pathways enriched in non-allergic compared to FA. In addition, overall pathway dissimilarities were found among different FAs. Conclusions: Our results demonstrate a link between IgE-mediated FA and the composition and metabolic activity of the gut microbiota.
Oral Immunotherapy for Food Allergy—a US Regulatory Perspective.
The recent approval of Palforzia for treatment of peanut allergy by the US Food and Drug Administration (USFDA) predicts that additional products for oral immunotherapy (OIT) are on the horizon. In this article, the authors review the legal framework by which the USFDA regulates products for OIT of food allergy and the clinical data that demonstrated that the safety and effectiveness profile of Palforzia supported approval and conclude with a discussion of oral food challenge (OFC) as a clinical endpoint to demonstrate safety and effectiveness of OIT products.
Impact of cold plasma processing on major peanut allergens.
Cold plasma is emerging as a novel food processing technology, with demonstrated efficacies for microbial inactivation and residual chemical dissipation of food products. Given the technology's multimodal action it has the potential to reduce allergens in foods, however data on the efficacy and mechanisms of action are sparse. This study investigates the efficacy of cold plasma on major peanut allergens (Ara h 1 and Ara h 2). For this purpose, dry, whole peanut (WP) and defatted peanut flour (DPF) were subjected to an atmospheric air discharge using a pin to plate cold plasma reactor for different treatment durations. With increases in plasma exposure, SDS-PAGE analysis revealed reduced protein solubility of the major peanut allergens. Alterations in allergenicity and structure of Ara h 1 and Ara h 2 were examined using ELISA and circular dichroism (CD) spectroscopy. Competitive ELISA with proteins purified from plasma treated WP or DPF revealed reduced antigenicity for both Ara h 1 and Ara h 2. The highest reduction in antigenicity was 65% for Ara h 1 and 66% Ara h 2 when purified from DPF. Results from CD spectroscopy analysis of purified proteins strongly suggests the reduction in antigenicity is due to modifications in the secondary structure of the allergens induced by plasma reactive species. Cold plasma is effective at reducing peanut protein solubility and causes changes in allergen structure leading to reduced antigenicity.
Two different composite markers predict severity and threshold dose in peanut allergy.
Background: Safe and cost-effective biological surrogate markers to evaluate the severity and threshold dose of peanut allergy (PA) reactions during an oral food challenge (OFC) are lacking. Objective: To evaluate biological markers associated with the severity and threshold dose of allergic reaction during OFC in a population of children with PA. Methods: Demographic and biologic parameters of children with peanut OFC and BAT results were collected. Patients were stratified into two severity groups (mild-to-moderate and severe) and two cumulative threshold dose groups: low (LCTG) ≤100mg crushed peanut; and high (HCTG) >100mg. Results: Among the 68 children included there was a 96% concordance between the OFC and BAT result for the diagnosis of PA. Of the 56 children with a positive OFC and BAT to peanut (median age: 8.8 years), severity of allergic reaction and cumulative threshold dose were not correlated (p=0.24). Higher Ara h 2-specific IgE and FcεRI-positive control values were both associated with severe reactions to peanut. Combining these two markers led to a 92% sensitivity [84%-97%] and 82% specificity [71%-89%] for severe reactions in all subjects. For children in the LCTG, a 4-variable composite marker including age, normalized basophil sensitivity (EC50), and FcεRI- and fMLP-positive control values, resulted in 97% sensitivity [89%-99%] and 61% specificity [49%-71%] CONCLUSION: Distinct composite markers including BAT allergen-specific and non-allergen-specific parameters appear to be associated with severity and cumulative threshold dose in children with PA.
A Prospective Validation of the NUT CRACKER Diagnostic Algorithm for Walnut and Pecan Allergy with Prediction of Severity.
Background: We previously devised an algorithm utilizing skin prick tests (SPT), basophil activation tests (BAT) and co-allergy status to reduce the need for oral food challenges (OFC) in 63 patients with suspected walnut/pecan allergies. Objective: To validate prospectively the NUT CRACKER diagnostic algorithm in a new cohort of patients (n=120) and to study the utility of SPT and BAT in predicting walnut-pecan allergy severity in both groups (n=183). Methods: Patients (n=183) aged 8 (6-11) years, median, (IQR) with suspected tree nut allergy were studied. SPT utilized ground nut extract (10 mg/ml) and BAT assessed allergen-induced basophil CD63 expression. Allergy was determined based on a recent reaction or a positive oral food challenge (OFC) and tolerance was defined by regular consumption or a negative OFC. Results: Walnut BAT was significantly higher in walnut/pecan dual compared to single walnut allergy (p=0.003) and predicted the need for epinephrine during positive walnut OFCs (p=0.009). Results of walnut and pecan-BAT correlated with the corresponding nut eliciting dose (p=0.014 and p<0.001, respectively). Receiver operating curves for walnut and pecan allergy, in the prospective cohort yielded area under the curves (AUC) ranging from 0.87-0.93 for SPT and BAT. Concordant with the original study, pecan allergic patients were all co-allergic for walnut. The algorithm decreased the need for OFCs by 78.8 % with 6/240 (2.5%) falsely positives and no false negatives.
Identification of Pru du 6 as a potential marker allergen for almond allergy.
Background: Oral food challenges have demonstrated that diagnosis of almond allergy based on extract-sIgE tests display low specificity. Molecular allergy diagnosis is expected to improve accuracy, but its value in diagnosing almond allergy remains unknown. Objective: To identify relevant almond allergens and examine their ability to improve almond allergy diagnosis. Methods: IgE-reactive proteins were purified from almond kernels. IgE-binding to almond extract and the allergens was analyzed by quantitative ELISA using sera from 18 subjects with a proven almond allergy. The control group consisted of sera from 18 subjects allergic to peanut and/or tree nuts but tolerant to almond. Results: Three IgE-binding proteins were identified: legumin (Pru du 6), alpha-hairpinin (Pru du 8) and mandelonitrile lyase (Pru du 10). Positive IgE (≥0.35 kU/L) to almond extract showed 94% sensitivity but only 33% specificity. IgE to Pru du 6 maintained high sensitivity (83%) and provided superior specificity (78%). Sera from almond-allergic subjects had significantly higher IgE levels to almond extract (P<0.0001) and Pru du 6 (P<0.0001) than sera from tolerant donors. Sensitization to Pru du 6 was highly specific for almond allergy, while frequencies of sensitization to legumins from peanut, walnut, hazelnut, and cashew were similar in both groups. IgE to Pru du 8 and Pru du 10 was less sensitive (41% and 67%), but showed specificities of 100% and 61%. Conclusion: The use of almond allergens markedly increases the diagnostic specificity compared to the extract. Pru du 6 is a potential new molecular marker for almond allergy.
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 is scarce. Objectives: To identify geographical differences in walnut component sensitization across Europe, explore co-sensitization 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; LTP 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 centres. 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/dog, positive SPT to walnut, and IgE to Jug r 1, 5, 7 or carbohydrate determinants (AUC = 0.81 [95%-CI 0.73-0.89]). Conclusions: Walnut allergic subjects across Europe show clear geographical differences in walnut component sensitization and co-sensitization 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.
Efficacy and safety of oral immunotherapy with AR101 in European children with a peanut allergy (ARTEMIS): a multicentre, double-blind, randomised, placebo-controlled phase 3 trial.
Background: Peanut allergy is the leading cause of food-related anaphylaxis. Current management options can negatively affect food allergy-related quality of life. We aimed to investigate the efficacy of an investigational oral biologic drug (AR101). Methods: The AR101 Trial in Europe Measuring Oral Immunotherapy Success in peanut-allergic children (ARTEMIS) trial was a multicentre, double-blind, randomised, placebo-controlled phase 3 trial done at 18 hospitals in Ireland, France, Germany, Italy, Spain, Sweden, and the UK. Children and adolescents with peanut allergy, aged 4-17 years, who developed dose-limiting symptoms to 300 mg or less peanut protein (equivalent to approximately one peanut kernel) during a double-blind placebo-controlled food challenge test at study entry were enrolled. Participants were randomly assigned (3:1) to receive daily doses of either AR101 oral immunotherapy (AR101 group) or a taste-masked placebo (placebo group). All participants, investigators, and care providers were masked to treatment allocation until the study was completed. Doses were increased every 2 weeks over 6 months until a dose of 300 mg was reached and maintained for 3 months. The primary endpoint was the proportion of participants in the intention-to-treat or safety population (defined as those participants who had been randomly assigned and had received at least one dose of the assigned drug) who could consume a single dose of 1000 mg (cumulative dose 2043 mg) peanut protein without developing dose-limiting allergic symptoms at an exit double-blind placebo-controlled food challenge after 9 months of treatment. Additional endpoints included safety (ie, the frequency and severity of adverse events) and changes in food allergy-related quality of life, assessed by use of age-appropriate Food Allergy Quality of Life Questionnaires (FAQLQs) and the Food Allergy Independent Measure (FAIM). The study is registered with ClinicalTrials.gov, NCT03201003, and is completed. Findings: Between June 12, 2017, and Feb 15, 2018, 227 patients were screened, of whom 175 were randomly assigned to the AR101 group (n=132) and the placebo group (n=43). All primary and secondary endpoints were met. 77 (58%) of 132 participants in the AR101 group tolerated 1000 mg peanut protein at the exit food challenge versus one (2%) of 43 participants in the placebo group (AR101-placebo treatment difference 56·0% [95% CI 44·1-65·2], p<0·0001). Adverse events were reported by almost all participants. The maximum severity of adverse events reported was mild or moderate for most participants who received AR101 (mild, 66 [50%] of 132 participants; moderate, 63 [48%]; and severe, one [1%]) or placebo (mild, 24 [56%] of 43 participants; moderate, 18 [42%]; severe, none). Participants aged 8-12 years in the AR101 group reported improvements that exceeded the minimum clinically important difference between the two groups across all FAQLQ domains. Additionally, participants in the AR101 group and their caregivers reported improvements that exceeded the minimum clinically important difference in FAIM domains related to the perceived likelihood and outcomes of a severe allergic reaction. Interpretation: AR101 oral immunotherapy treatment led to rapid desensitisation to peanut protein, with a predictable safety profile that improved with treatment, and an associated improvement in self-reported and caregiver-reported food allergy-related quality of life. These patient-oriented outcomes provide invaluable data to help physicians, patients, and caregivers make informed, shared decisions on the management of peanut allergy. Funding: Aimmune Therapeutics.