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In this work, a novel magnetic beads (MBs)-based immunosensing approach for the rapid and simultaneous determination of the main peanut allergenic proteins (Ara h 1 and Ara h 2) is reported. It involves the use of sandwich-type immunoassays using selective capture and detector antibodies and carboxylic acid-modified magnetic beads (HOOC-MBs). Amperometric detection at −0.20 V was performed using dual screen-printed carbon electrodes (SPdCEs) and the H2O2/hydroquinone (HQ) system. This methodology exhibits high sensitivity and selectivity for the target proteins providing detection limits of 18.0 and 0.07 ng/mL for Ara h 1 and Ara h 2, respectively, with an assay time of only 2 h. The usefulness of the approach was evaluated by detecting the endogenous content of both allergenic proteins in different food extracts as well as trace amounts of peanut allergen (0.0001% or 1.0 mg/kg) in wheat flour spiked samples. The developed platform provides better Low detection limits (LODs) in shorter assay times than those claimed for the allergen specific commercial ELISA kits using the same immunoreagents and quantitative information on individual food allergen levels. Moreover, the flexibility of the methodology makes it readily translate to the detection of other food-allergens.

Background Threshold levels for peanut allergy determined by using oral challenges are important for the food industry with regard to allergen labeling. Moreover, the utility of biological markers in predicting threshold levels is uncertain. Objective We sought to use a modified oral food challenge regimen that might determine threshold levels for peanut allergy mimicking a more real-life exposure and to correlate the eliciting dose (ED) and severity of clinical reaction in children with peanut allergy with B-cell, T-cell, and effector cell markers. Methods A modified food challenge procedure with doses scheduled 2 hours apart was used in 63 children with peanut allergy. All children received a maximum of 8 semi-log increasing titration steps of roasted peanuts ranging from 3 to 4500 mg of peanut protein until objective allergic reactions occurred. Severity of symptoms was graded from I to V. Biological markers were measured before challenge. Results Forty-five of 63 patients showed objective symptoms after greater than 30 minutes, with a median latency of clinical reaction of 55 minutes. By using a log-normal dose-distribution model, the ED5 was calculated to be 1.95 mg of peanut protein. The ED was significantly and inversely correlated with peanut- and Ara h 2-specific IgE levels, skin prick test responses, basophil activation, and TH2 cytokine production by PBMCs. Symptom severity did not correlate with any of the markers or the ED. Conclusion This modified food challenge procedure might better reflect threshold levels for peanut allergy than the standard procedure because most of the patients reacted at a time interval of greater than 30 minutes. By using this model, threshold levels, but not severity, could be correlated with biological markers. [ABSTRACT FROM AUTHOR]

Simplified methods to purify major peanut allergens (Ara h 1, Ara h 2, and Ara h 3) were investigated to facilitate current laborious and time-consuming conventional purification methods, the combination of ammonium sulfate fractionation (ASF) and successive column chromatographic separations. Crude peanut proteins obtained from defatted peanut were subjected to ASF at higher concentrations of ammonium sulfate than those in conventional methods and then dialyzed against water. Purified Ara h 1, Ara h 2, and Ara h 3 were obtained by ultrafiltration(s) of the dialyzed supernatant in ASF (80 - 90%), the dialyzed precipitate in ASF (40 - 50%), and the dialyzed precipitate in ASF (0 - 40%), respectively. The purities of Ara h 1, Ara h 2, and Ara h 3 were 91.3%, 86.4%, and 93.9%, respectively. The simplified methods enable the purification of the three major peanut allergens in a relatively short time without the use of chromatographic separation techniques. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two E-PB sensors were designed for detection of anti-Ara h 2 antibodies. [•] A sensor was fabricated with a His-tagged peptide probe on a Ni(II) NTA monolayer. [•] A sensor was fabricated using the conventional method with a thiolated probe. [•] The His-tagged sensor has a lower detection limit than the thiolated sensor. [•] Both sensors are regenerable and reusable. [ABSTRACT FROM AUTHOR]

Peanut seeds are currently widely used as source of human food ingredients in the United States of America and in European countries due to their high quality protein and oil content. This article describes the classification and molecular biology of peanut seed allergens with particular reference to their cross-reactivities. Currently, the IUIS allergen nomenclature subcommittee accepts 12 peanut allergens. Two allergens belong to the cupin and four to the prolamin superfamily, and six are distributed among profilins, Bet v 1-like proteins, oleosins, and defensins. Clinical observations frequently report an association of peanut allergy with allergies to legumes, tree nuts, seeds, fruits and pollen. Molecular cross-reactivity has been described between members of the Bet v 1-like proteins, the non-specific lipid transfer proteins, and the profilins. This review also addresses the less well-studied cross-reactivity between cupin and prolamin allergens of peanuts and of other plant food sources and the recently discovered cross-reactivity between peanut allergens of unrelated protein families. [ABSTRACT FROM AUTHOR]

Highlights: [•] The study provides an approach to reduce allergenic proteins in peanut product. [•] A sequential process of ultrasonication followed by enzyme digestion was developed. [•] Ultrasound assisted protease digestion greatly increased peanut protein solubility. [•] Ultrasound–enzyme combination significantly lowered Ara h 1 and Ara h 2 in peanuts. [•] Ultrasound–enzyme combination significantly lowered IgE binding of peanut extract. [ABSTRACT FROM AUTHOR]

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut. [ABSTRACT FROM AUTHOR]

Food allergy has become a major public health concern in westernized countries, and allergic reactions to peanuts are particularly common and severe. Allergens are defined as antigens that elicit an IgE response, and most allergenic materials (e.g., pollens, danders, and foods) contain multiple allergenic proteins. This has led to the concept that there are 'major' allergens and allergens of less importance. 'Major allergens' have been defined as allergens that bind a large amount of IgE from the majority of patients and have biologic activity. However, the ability of an allergen to cross-link complexes of IgE and its high-affinity receptor FcεRI (IgE/FcεRI), which we have termed its allergic effector activity, does not correlate well with assays of IgE binding. To identify the proteins that are the most active allergens in peanuts, we and others have employed in vitro model assays of allergen-mediated cross-linking of IgE/FcεRI complexes and have demonstrated that the most potent allergens are not necessarily those that bind the most IgE. The importance of a specific allergen can be determined by measuring the allergic effector activity of that allergen following purification under non-denaturing conditions and by specifically removing the allergen from a complex allergenic extract either by chromatography or by specific immunodepletion. In our studies of peanut allergens, our laboratory has found that two related allergens, Ara h 2 and Ara h 6, together account for the majority of the effector activity in a crude peanut extract. Furthermore, murine studies demonstrated that Ara h 2 and Ara h 6 are not only the major elicitors of anaphylaxis in this system, but also can effectively desensitize peanut-allergic mice. As a result of these observations, we propose that the definition of a major allergen should be based on the potency of that allergen in assays of allergic effector activity and demonstration that removal of that allergen from an extract results in loss of potency. Using these criteria, Ara h 2 and Ara h 6 are the major peanut allergens. [ABSTRACT FROM AUTHOR]

Introduction As replacement for the oral food challenge, decision-points for sensitization test have been established, but suboptimal sensitivity and/or specificity, as well as regional differences, have reduced the clinical usability. IgE toward specific peanut protein components has been reported to be of value, but data on correlation with clinical data are sparse. Our aim was to correlate IgE values with the outcome of peanut challenges. Method Data from 175 positive and 30 negative peanut challenges in patients aged 1-26 years were retrospectively correlated with the levels of specific IgE to peanut and peanut components (Ara h 1-3, h 8, and h 9). Result The best correlation between IgE and clinical thresholds was found for Ara h 2 (ρs = −0.30, P < 0.01). A cutoff of Ara h 2 > 1.63 kU/l yielded a specificity = 1.00, with a corresponding sensitivity of 0.70. Symptom severity elicited during challenge correlated significantly with the levels of Ara h 2 (ρs = 0.60, P < 0.0001), but large individual variation was found. Conclusion The level of IgE toward Ara h 2 can improve diagnostic accuracy by introducing a more clear-cut decision-point with an optimal specificity maintaining a high sensitivity. In our study, this would have reduced the necessary number of challenges to be performed from 205 to 92. Extrapolation between centers is difficult and decision-points need to be addressed in relation to settings and population. Further component-resolved diagnostic cannot replace oral challenge neither in determining thresholds nor in the assessment of severity of symptoms elicited during challenge. [ABSTRACT FROM AUTHOR]

Background: The moderately homologous (approx. 60%) proteins Ara h 2 and Ara h 6 are the most potent peanut al-lergens. This study was designed to define the relative indi-vidual contributions of Ara h 2 and Ara h 6 to the overall al-lergenic activity of a crude peanut extract (CPE). Methods: Ara h 2 and Ara h 6 were removed from CPE by gel filtration chromatography. Ara h 2.01, Ara h 2.02 and Ara h 6 were fur-ther purified (>99%). The potency of each allergen and the ability of these allergens to reconstitute the allergenic activ-ity of CPE depleted of Ara h 2 and Ara h 6 was measured with RBL SX-38 cells sensitized with IgE from sensitized peanut al-lergic patients. Results: The potency of the native proteins were significantly different (p < 0.0001) although not dra-matically so, with a rank order of Ara h 2.01 > Ara h 2.02 > Ara h 6. The addition of either purified Ara h 2 or Ara h 6 inde-pendently at their original concentration to CPE depleted of both Ara h 2 and Ara h 6 restored 80-100% of the original CPE allergenic activity. Addition of both Ara h 2 and Ara h 6 consistently completely restored the allergenic activity of CPE. Conclusions: These studies indicate that either Ara h 2 or Ara h 6 independently can account for most of the aller-genic activity in a CPE and demonstrate important redun-dancy in the allergenic activity of these related molecules. [ABSTRACT FROM AUTHOR]

Background Despite the frequency and severity of peanut allergy, the only approved treatment is strict avoidance. Different types of immunotherapy with crude peanut extract are not universally effective and have been associated with relatively high adverse reaction rates. Objective We sought to determine whether in silico predictive algorithms were useful in identifying candidate peptides for an Ara h 2 peptide-based vaccine using peanut-allergic patients' peripheral blood mononuclear cells ( PBMCs) in vitro. A human leucocyte antigen ( HLA) distribution analysis was also performed. Methods Major histocompatibility complex ( MHC)-class II-binding peptides were predicted using Net MHCIIpan-2.0 and Net MHCII-2.2 algorithms. PBMCs from 80 peanut-allergic patients were stimulated with overlapping 20-mer Ara h 2 peptides. Cell supernatant cytokine profiles were evaluated by multiplex assays. HLA- DRB1* and HLA- DQB1* typing were performed. Results Four regions of overlapping sequences induced PBMC proliferation and predominant Th2 cytokine production. HLA genotyping showed 30 different DRB1* allele specificities and eight DQ serological specificities. The in silico analysis revealed similar relevant regions and predicted identical or similar core 9-mer epitopes to those identified in vitro. If relevant peptides, as determined by either in vitro or in silico analysis (15 peptides and 9 core epitopes respectively), were used in a peptide-based vaccine, they would cover virtually all subjects in the cohort studied. Conclusions and Clinical Relevance Four dominant regions in Ara h 2 have been identified, containing sequences that could serve as potential candidates for peptide-based immunotherapy. MHC-class II-based T cell epitope prediction algorithms for HLA- DR and - DQ loci accurately predicted Ara h 2 T cell epitopes in peanut-allergic subjects, suggesting their potential utility in a peptide-based vaccine design for food allergy. [ABSTRACT FROM AUTHOR]

Background: A diagnostic prediction model for peanut allergy in children was recently published, using 6 predictors: sex, age, history, skin prick test, peanut specific immunoglobulin E (sIgE), and total IgE minus peanut sIgE. Objectives: To validate this model and update it by adding allergic rhinitis, atopic dermatitis, and sIgE to peanut components Ara h 1, 2, 3, and 8 as candidate predictors. To develop a new model based only on sIgE to peanut components. Methods: Validation was performed by testing discrimination (diagnostic value) with an area under the receiver operating characteristic curve and calibration (agreement between predicted and observed frequencies of peanut allergy) with the Hosmer-Lemeshow test and a calibration plot. The performance of the (updated) models was similarly analyzed. Results: Validation of the model in 100 patients showed good discrimination (88%) but poor calibration (P < .001). In the updating process, age, history, and additional candidate predictors did not significantly increase discrimination, being 94%, and leaving only 4 predictors of the original model: sex, skin prick test, peanut sIgE, and total IgE minus sIgE. When building a model with sIgE to peanut components, Ara h 2 was the only predictor, with a discriminative ability of 90%. Cutoff values with 100% positive and negative predictive values could be calculated for both the updated model and sIgE to Ara h 2. In this way, the outcome of the food challenge could be predicted with 100% accuracy in 59% (updated model) and 50% (Ara h 2) of the patients. Conclusions: Discrimination of the validated model was good; however, calibration was poor. The discriminative ability of Ara h 2 was almost comparable to that of the updated model, containing 4 predictors. With both models, the need for peanut challenges could be reduced by at least 50%. [ABSTRACT FROM AUTHOR]

The recently published reference genome of peanuts enables a detailed molecular description of the allergenic proteins of the seed. We used LC-MS/MS to investigate peanuts of different genotypes to assess variability and to better describe naturally occurring allergens and isoforms. Using relative quantification by mass spectrometry, minor variation of some allergenic proteins was observed, but total levels of Ara h 1, 2, 3, and 6 were relatively consistent among 20 genotypes. Previously published RP-HPLC methodology was used for comparison. The abundance of three Ara h 3 isoforms were variable among the genotypes and contributed to a large proportion of total Ara h 3 where present. Previously unpublished hydroxyproline sites were identified in Ara h 1 and 3. Hydroxylation did not vary significantly where sites were present. Peanut allergen composition was largely stable, with only some isoforms displaying differences between genotypes. The resulting differences in allergenicity are of unknown clinical significance but are likely to be minor. The data presented herein allow for the design of targeted MS methodology to allow the quantitation and therefore control of peanut allergens of clinical relevance and observed variability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marsh, Palmer, Koppelman and Johnson.)

Background There are no available clinical tests that can accurately predict peanut allergy ( PA) and/or anaphylaxis. This study is aimed at evaluating whether the component-resolved diagnostic ( CRD) Ig E and Ig G4 tests can (i) distinguish PA from asymptomatic peanut sensitization ( PS) and (ii) differentiate anaphylactic from nonanaphylactic PA. Methods This study included 20 nonatopic controls, 58 asymptomatically peanut-sensitized children, 55 nonanaphylactic, and 53 anaphylactic PA cases from the Chicago Food Allergy Study. IgE and IgG4 to 103 allergens were measured using the Immuno CAP ISAC technology and were compared among each group of children. The random forest test was applied to estimate each allergen's ability to predict PA and/or peanut anaphylaxis. Results Peanut allergy cases (with or without anaphylaxis) had significantly higher Ig E reactivity to Ara h 1-3 (peanut allergens) and Gly m 5-6 (soy allergens) than asymptomatically sensitized children ( P < 0.00001). Similar but more modest relationships were found for Ig G4 to Ara h 2 ( P < 0.01). Ig E to Ara h 2 was the major contributor to accurate discrimination between PA and asymptomatic sensitization. With an optimal cutoff point of 0.65 ISU- E, it conferred 99.1% sensitivity, 98.3% specificity, and a 1.2% misclassification rate in the prediction of PA, which represented a higher discriminative accuracy than Ig E to whole peanut extract ( P = 0.008). However, none of the Ig E and/or Ig G4 tests could significantly differentiate peanut anaphylaxis from nonanaphylactic PA. Conclusions Ig E to Ara h 2 can efficiently differentiate clinical PA from asymptomatic PS, which may represent a major step forward in the diagnosis of PA. [ABSTRACT FROM AUTHOR]

Background: Seed storage proteins (SSP; Ara h 1, Ara h 2, Ara h 3) have been shown to be major peanut allergens, although recently, peanut lipid transfer protein has been reported to be an important allergen in the Mediterranean area. We sought to investigate the sensitization pattern to peanut SSP and vegetable pan-allergens in a group of peanut-allergic children compared with a peanut-tolerant group. Methods: One hundred and twenty-three children who presented with food allergy were included in the study. Tolerance to peanut ingestion was assessed. Specific IgE was determined by ImmunoCAP, and microarray ISAC was performed. Sensitization frequencies and levels of specific IgE were compared between groups. Results: Fifty-five of 123 children presented symptoms upon contact or ingestion. Frequency of sensitization to Ara h 1, Ara h 2, and Ara h 3 was 60.0%, 72.7%, and 43.6%, respectively, in the group of allergic children vs. 7.4%, 1.5%, and 7.4% in the group of tolerant children. Levels of specific IgE against Ara h 1, Ara h 2, and Ara h 3 were significantly higher in the allergic group (p < 0.001). The frequency of sensitization and the levels of specific IgE against Cor a 8 (36.4% vs. 16.2%) were significantly higher in the allergic children, whereas no significant differences were found for Pru p 3. No differences were seen for other pan-allergens. Patients sensitized to SSP, regardless of sensitization to nsLTP, were allergic rather than tolerant. Conclusion: In our population, peanut-allergic children were mainly sensitive to SSP. A few patients were also sensitive to some nsLTPs. No differences were shown in other pan-allergens. [ABSTRACT FROM AUTHOR]

Peanut allergy represents one of the most severe IgE-mediated reactions with food, but to date, the only effective way to prevent peanut allergy is total avoidance. If allergens could be mitigated during food processing before a product reaches the consumer, this would substantially lessen the food allergy problem. The efficacy of pulsed ultraviolet light (PUV), a novel food processing technology, on reducing peanut allergens, was examined. This study revealed for the first time that PUV was also capable of deactivating Ara h 2, the most potent allergenic protein of peanut. Protein extracts from raw and roasted peanuts were treated for 2, 4, and 6 min and peanut butter slurry was treated for 1, 2, and 3 min in a Xenon Steripulse XL 3000® PUV system. The distance from the central axis of the lamp was varied at 10.8, 14.6, and 18.2 cm. The SDS-PAGE showed a reduction in the protein band intensity for Ara h 1, Ara h 2, and Ara h 3 at the energy levels ranging from 111.6 to 223.2 J/cm. Reduction of the protein band intensity for peanut allergens increased with treatment time but decreased with increased distance from the PUV lamp. The ELISA for peanut extracts and peanut butter slurry showed a reduction in IgE binding of up to 12.9- and 6.7-folds, respectively, compared to control. [ABSTRACT FROM AUTHOR]

Diagnosis of peanut allergy has improved thanks to component-resolved diagnostics. Peanut allergen component Ara h 2 is considered to indicate true peanut allergy. The component Ara h 6 is structurally similar to Ara h 2, but the diagnostic value of analyzing IgE antibodies to Ara h 6 is unclear. A boy sensitized (≥0.35 kUA/l) to Ara h 8 but not to Ara h 1, Ara h 2 and Ara h 3 was challenged with peanut and developed grade II anaphylaxis. In serum collected at the time of challenge a doubling of IgE to the peanut allergen extract was observed compared to allergy testing 9 months earlier. In contrast, IgE levels to Ara h 1, Ara h 2, Ara h 3 and to Ara h 8 were rather unchanged. After another 2 months, Ara h 6 was analyzed and revealed a level of 24 kUA/l whilst Ara h 2 was 0.12 kUA/l. We suggest that IgE sensitization to Ara h 6 caused the reaction and conclude that analyses of IgE levels to peanut and peanut components should be performed in connection with a challenge. Furthermore, levels to Ara h 2 below 0.35 kUA/l may still indicate a risk of severe reaction at the time of challenge since in rare cases, Ara h 6 IgE antibodies may be present without occurrence of IgE antibodies to Ara h 2. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Isolated Ara h 8 sensitization is suggested to be associated with no or mild symptoms among peanut-sensitized subjects. Objective: We sought to investigate the occurrence of systemic reactions in children with isolated sensitization to Ara h 8. Methods: Participants were 144 children sensitized to Ara h 8 (≥0.35 kUA/L) but not to Ara h 1, Ara h 2, or Ara h 3 (<0.35 kUA/L). An open oral challenge with peanut was performed in those subjects who did not consume peanut regularly, and an extended IgE reactivity profile was obtained. If the child had a documented history of systemic reactions up to grade I anaphylaxis, double-blind, placebo-controlled food challenges were performed. Results: One hundred twenty-nine (89.5%) children were either peanut consumers or did not react to peanut challenge. Another 14 (9.7%) children experienced oral cavity symptoms at the first 2 but not subsequent challenge doses. At the time of the double-blind, placebo-controlled food challenge, 1 boy with a previous mild systemic reaction to peanut experienced lip swelling, stomach cramping, and objective tiredness. Reanalysis of IgE levels showed an increase in peanut IgE levels from 1.5 to 8.8 kUA/L, but IgE levels to Ara h 8 remained stable and IgE levels to Ara h 1, Ara h 2, and Ara h 3 were all still less than 0.35 kUA/L. The IgE level to Ara h 6 was 0.45 kUA/L. Conclusion: Isolated Ara h 8 sensitization indicates tolerance to peanuts in almost all cases. However, sensitization against thus far unidentified determinants in peanut might cause symptoms in rare cases. [ABSTRACT FROM AUTHOR]

To cite this article: Sverremark-Ekström E, Hultgren EH, Borres MP, Nilsson C. Peanut sensitization during the first 5 yr of life is associated with elevated levels of peanut-specific IgG. Pediatr Allergy Immunol 2012: 23: 224-229. Abstract Background: Allergen-specific IgE antibodies are implicated in allergic diseases while allergen-specific IgG antibodies have been proposed to prevent allergic reactions. The objective for this study was to study whether the immune response (IgG and IgG4) to peanut differs in IgE-sensitized and non-sensitized young children. Methods: A total of 239 children have been followed prospectively from birth to 5 yr of age. The levels of IgG and IgG4 to peanut, Ara h 2, and Ara h 8 were analyzed at 2 and 5 yr of age and related to IgE sensitization and peanut consumption. Results: The levels of peanut-specific IgG and IgG4 were significantly higher in peanut-sensitized children at 2 and 5 yr of age when compared with non-sensitized children and children sensitized to other food/inhalant allergens. A strong correlation was seen between levels of peanut-specific IgG/IgG4-ratios and peanut-specific IgE at 5 yr of age. Children avoiding peanuts, a subgroup of the peanut sensitized, had statistically significant higher levels of IgE to peanut and a tendency of higher IgG and IgG4 levels to peanut. In the avoidance group, significant correlations between IgE and IgG/IgG4 to peanut were found compared with children eating peanuts. Conclusion: Peanut-specific IgG or IgG4 levels were elevated in peanut-sensitized children especially those avoiding peanuts. In our study, IgG and IgG4 do not seem to indicate tolerance or protection from sensitization. [ABSTRACT FROM AUTHOR]

Background: Measurement of whole peanut-specific IgE (sIgE) is often used to confirm sensitization but does not reliably predict allergy. Ara h 2 is the dominant peanut allergen detected in 90% to 100% of patients with peanut allergy and could help improve diagnosis. Objectives: We sought to determine whether Ara h 2 testing might improve the accuracy of diagnosing peanut allergy and therefore circumvent the need for an oral food challenge (OFC). Methods: Infants from the population-based HealthNuts study underwent skin prick tests to determine peanut sensitization and subsequently underwent a peanut OFC to confirm allergy status. In a stratified random sample of 200 infants (100 with peanut allergy and 100 with peanut tolerance), whole peanut sIgE and Ara h 2 sIgE levels were quantified by using fluorescence enzyme immunoassay. Results: By using the previously published 95% positive predictive value of 15 kUA/L for whole peanut sIgE, a corresponding specificity of 98% (95% CI, 93% to 100%) was found in this study cohort. At the equivalent specificity of 98%, the sensitivity of Ara h 2 sIgE is 60% (95% CI, 50% to 70%), correctly identifying 60% of subjects with true peanut allergy compared with only 26% correctly identified by using whole peanut sIgE. We report that when using a combined approach of plasma sIgE testing for whole peanut followed by Ara h 2 for the diagnosis of peanut allergy, the number of OFCs required is reduced by almost two thirds. Conclusion: Ara h 2 plasma sIgE test levels provide higher diagnostic accuracy than whole peanut plasma sIgE levels and could be considered a new diagnostic tool to distinguish peanut allergy from peanut tolerance, which might reduce the need for an OFC. [Copyright &y& Elsevier]

Background: Peanut may cause severe reactions in allergic individuals. The objective was to evaluate IgE antibodies to various recombinant (r) peanut and birch pollen allergens in relation to IgE levels to whole peanut extract and severe allergic reactions to peanut. Methods: Seventy-four Swedish peanut-allergic patients (age: 14-61 years) reported previous peanut exposure and associated symptoms using a questionnaire. Their IgE reactivity to peanut, birch pollen and individual allergen components was analyzed using ImmunoCAP®. Results: Of the 48 subjects sensitized to Ara h 1, 2 or 3, 60% had peanut-specific IgE levels >15 kUA/l, while 100% of the subjects without detectable IgE to these allergens had low peanut-specific IgE levels (<10 kUA/l). The levels of IgE to rAra h 8, rBet v 1 and birch pollen were highly correlated (rS = 0.94, p < 0.0001). Fifty-eight patients reported adverse reactions after accidental or deliberate peanut exposure (oral, inhalation or skin) of whom 41 had IgE to rAra h 1, 2 or 3. Symptoms of respiratory distress were associated with sensitization to Ara h 1, 2 or 3 (56 vs. 18%, p < 0.01). Two cases of anaphylaxis were reported among the individuals sensitized to Ara h 1-3. IgE to rAra h 8, rAra h 9, profilin or cross-reactive carbohydrate determinants were not associated with severe symptoms. Conclusions: The results indicate that IgE reactivity to Ara h 1, 2 and 3 is associated with severe reactions after exposure to peanut in Swedish patients. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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Abstract: It is important to know the contribution of specific allergens to a complex allergenic extract and to have a dependable method to assess the effector activity of an extract specifically depleted of that allergen. We have previously shown that removal of the major peanut allergen, Ara h 2, from a crude peanut extract (CPE) minimally altered the effector activity of the extract. Here we describe in detail the methodology used to generate specific rabbit anti-peptide antibodies to remove a related peanut allergen, Ara h 6, from CPE and describe an improvement in the RBL SX-38 cell assay used to assess the effector activity of treated extracts. Our results show that although Ara h 2 and Ara h 6 can be selectively removed from a CPE, removal of each alone from a CPE had no significant effect on the effector activity. However, removal of Ara h 2 and Ara h 6 together significantly reduced the effector activity of CPE. [Copyright &y& Elsevier]

Abstract: This study investigated the use of enzymatic treatment to reduce peanut allergens in peanut kernels as affected by processing conditions. Two major peanut allergens, Ara h 1 and Ara h 2, were used as indicators of process effectiveness. Enzymatic treatment effectively reduced Ara h 1 and Ara h 2 in roasted peanut kernels by up to 100% under optimal conditions. For instance, treatment of roasted peanut kernels with α-chymotrypsin and trypsin for 1–3h significantly increased the solubility of peanut protein while reducing Ara h 1 and Ara h 2 in peanut kernel extracts by 100% and 98%, respectively, based on ELISA readings. Ara h 1 and Ara h 2 levels in peanut protein extracts were inversely correlated with protein solubility in roasted peanut. Blanching of kernels enhanced the effectiveness of enzyme treatment in roasted peanuts but not in raw peanuts. The optimal concentration of enzyme was determined by response surface to be in the range of 0.1–0.2%. No consistent results were obtained for raw peanut kernels since Ara h 1 and Ara h 2 increased in peanut protein extracts under some treatment conditions and decreased in others. [Copyright &y& Elsevier]

Background: Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80–90% of peanut allergic patients. Methods: The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 A ˚ resolution using a customized maltose-binding protein (MBP)-fusion system. IgE antibody binding to the MBP fusion construct vs the natural allergen was compared by ELISA using sera from peanut allergic patients. Results: The structure of Ara h 2 is a five-helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP–Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Subpopulation 1 (n = 14) showed an excellent correlation of IgE antibody binding to natural vs recombinant Ara h 2. Subpopulation 2 (n = 15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in subpopulation 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct. Discussion: The reduced IgE binding to the MBP–Ara h 2 of subpopulation 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP–Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy. [ABSTRACT FROM AUTHOR]

Chiang WC, Pons L, Kidon MI, Liew WK, Goh A, Wesley Burks A. Serological and clinical characteristics of children with peanut sensitization in an Asian community. Pediatr Allergy Immunol 2010: 21: e429–e438. © 2009 John Wiley & Sons A/S In the past two decades, peanut allergy prevalence has increased in the West but has been perceived as having remained low in Asia. To review the clinical presentation of Asian children with peanut hypersensitivity and measure their IgE responses to major peanut allergens. We enrolled 31 children presenting with various allergies and a positive skin prick test to peanut from the Children’s hospital outpatient allergy clinic in Singapore. A detailed questionnaire was completed by parents. The children’s serum IgE specific to native Ara h 1, native Ara h 2, and recombinant Ara h 3 were detected using ELISA. Of the 31 patients, 19 had previously documented reactions to peanuts, while 12 had no previous clinical reaction. Most, 89.5% (17/19) of first reactions featured skin changes (urticaria, erythema, angioedema), but only 36.8% (7/19) involved skin symptoms alone. Respiratory symptoms and GI symptoms occurred in 42.1% and 26.3% of patients respectively and did not occur as the sole manifestation of reaction. The most common GI manifestation was emesis, present in 26.3% (5/19) of subjects. Two children experienced impaired consciousness with systemic, anaphylactic events. Although most sought treatment for their first peanut reaction only one patient received epinephrine. Half of our patients reported a subsequent accidental ingestion after the diagnosis of peanut allergy, with a median time from diagnosis to first accidental ingestion of 4 months and a reported increased severity of reaction in approximately half of the repeat exposures. Eighty-seven percent of children had specific IgE directed against at least one of the major peanut allergens. Among all patients, 87.1% had IgE specific to both Ara h 1 and Ara h 2 and 54.8% to rAra h 3. Asian children with peanut sensitization have clinically similar presentations and respond to the same major allergenic proteins as their Western counterparts. The perceived differences between the populations in this context do not stem from divergent clinical or immunological responses. [ABSTRACT FROM AUTHOR]

The role of antibody avidity in allergy is poorly understood and there is no existing literature describing antibody avidity in food allergy. The main aim of this study was to investigate IgE and IgG avidity to a total peanut protein extract (TPPE) and purified Ara h 2 in a group of well-characterized peanut allergic individuals. Forty peanut allergic patients underwent a double-blind placebo-controlled low-dose peanut challenge, during which the severity of the patients’ peanut allergy was scored. Serum peanut-specific IgE (psIgE) and IgG (psIgG) concentrations were measured for 37 individuals and the avidities of the same antibodies to a TPPE and purified Ara h 2 were determined using a thiocyanate ELISA method. Both IgE and IgG avidity to Ara h 2 showed weak positive correlations with challenge score [r = 0.459 (p = 0.012) and r = 0.486 (p = 0.003), respectively]. IgE avidity to TPPE showed a weak positive correlation with skin prick test results (SPT), r = 0.467 (p = 0.004) and there was an inverse relationship between the ratio of total IgE:psIgE and challenge score r = −0.561 (p < 0.001). No significant relationship was found between the ratios of IgE avidity:IgG avidity and challenge score or SPT. This is the first description of IgE and IgG avidity in peanut allergy, and it appears that the avidities of IgE and IgG antibodies to purified Ara h 2 are weakly related to the severity of peanut allergy (as measured by a challenge score). [ABSTRACT FROM AUTHOR]

Background Ara h 2 is a potent peanut allergen but its contribution to the ability of a crude peanut extract (CPE) to cross-link IgE and activate mast cells has not been rigorously evaluated. Objective To measure the contribution that Ara h 2 makes to the effector function of a CPE. Methods Ara h 2 was specifically removed from a CPE as demonstrated by immunoblots, 2D gels, and an inhibitory ELISA. Functional assays of sham-treated and Ara h 2-depleted CPEs were performed with RBL SX-38 cells sensitized with IgE from highly peanut-allergic subjects and with naturally sensitized basophils. Results Depletion of ∼99% of the Ara h 2 from the CPE led to an increase in the concentration of the CPE necessary to give 50% of maximal degranulation (EC50) of the SX-38 cells following sensitization with sera that contain anti-Ara h 2 IgE. Assays with a pool of 10 sera showed a small but significant increase in the EC50 following depletion of Ara h 2 (1.65±0.15-fold; P<0.05) and assays of seven individual sera showed a similar increase in the average EC50 (1.7±0.2-fold; P<0.02). The percent of the anti-peanut IgE that binds Ara h 2 correlated with an increase in the EC50 of the CPE following depletion of Ara h 2 ( r=0.83; P<0.02). On the other hand, data from three of these patients studied with a basophil histamine release assay did not show a significant effect of depletion of Ara h 2. Conclusion Based on its ability to cross-link IgE effectively, Ara h 2 is clearly an important peanut allergen. Its ability to cross-link IgE effectively from a specific serum is related to the proportion of anti-Ara h 2 in that serum but Ara h 2 does not account for a majority of the effector activity of the CPE for any of the sera studied. [ABSTRACT FROM AUTHOR]

Abstract: Peanut allergy is one of the most serious food allergies. The major peanut allergens are designated Ara h 1, Ara h 2 and Ara h 3. A previously unidentified ara h 3-related cDNA, ara h 3-im, was isolated and its corresponding protein product confirmed in peanut seed by N-terminal sequencing. Ara h 3-im is distinct from other related allergens in having a novel N-terminal sequence, lacking two potential N-glycosylation sites, missing most of the fourth IgE-binding epitope found in Ara h 3, and having a different predicted three-dimensional structure. Immunoblotting showed that the 36kDa Ara h 3-im polypeptide was not recognized by IgE from the sera of peanut sensitive patients that recognized other major peanut allergen polypeptides. ara h 3-im is a new peanut arachin gene that encodes an expressed protein in peanut seed, Ara h 3-im, that shares features with other 11S globulin genes. Initial results indicate that Ara h 3-im has potentially lower allergenic properties than previously characterized peanut allergens which may aid in the production of a hypoallergenic peanut. [Copyright &y& Elsevier]

Abstract: Allergy to peanut and tree nuts is characterised by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Although peanut is the most common cause of nut allergy, peanut allergic patients are frequently also sensitive to tree nuts. It is not known if this is due to cross-reactivity between peanut and tree nut allergens. In this study, the major peanut allergen Ara h 2 was cloned from peanut cDNA, expressed in E. coli cells as a His-tag fusion protein and purified using a Ni-NTA column. Immunoblotting, ELISA and basophil activation indicated by CD63 expression all confirmed the IgE reactivity and biological activity of rAra h 2. To determine whether or not this allergen plays a role in IgE cross-reactivity between peanut and tree nuts, inhibition ELISA was performed. Pre-incubation of serum from peanut allergic patients with increasing concentrations of almond or Brazil nut extract inhibited IgE binding to rAra h 2. Purified rAra h 2-specific serum IgE antibodies also bound to proteins present in almond and Brazil nut extracts by immunoblotting. This indicates that the major peanut allergen, Ara h 2, shares common IgE-binding epitopes with almond and Brazil nut allergens, which may contribute to the high incidence of tree nut sensitisation in peanut allergic individuals. [Copyright &y& Elsevier]

Background Recognition of specific peanut allergens or the diversity of IgE binding to peanut allergens may play a role in the elicitation of severe allergic reactions. Objective To investigate whether sensitization to individual allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 is correlated with clinical severity. Methods The reactivity of purified peanut allergens was measured by skin prick test (SPT) and by IgE immunoblot in 30 patients. The results were related to the clinical reactivity by history, and in 25 of them to the eliciting dose (ED). Results The majority of patients recognized Ara h 2 and Ara h 6. Patients with severe symptoms had a higher SPT response to Ara h 2 and Ara h 6 at low concentrations (0.1 μg/mL) and to Ara h 1 and Ara h 3 at higher concentrations (100 μg/mL), compared with patients with mild symptoms. They also recognized a greater number of allergens and showed a higher cumulative SPT response compared with patients with mild symptoms. No significant differences were observed between patients with a low or high ED. Conclusions Ara h 2 and Ara h 6 appeared to be more potent than Ara h 1 and Ara h 3. Both SPT reactivity to low concentrations of Ara h 2 and Ara h 6 and to higher concentrations of Ara h 1 and Ara h 3 were shown to be indicative of severe symptoms. [ABSTRACT FROM AUTHOR]

Background: Detailed assessment of antibody responses to allergens reveals clinically relevant information about both host response and antigen structure. Microarray technology offers advantages of scale and parallel design over previous methods of epitope mapping. Objective: We designed a redundant peptide microarray for IgE and IgG4 epitope mapping of the previously characterized peanut allergen, Ara h 2. Methods: Six complete sets of overlapping peptides were commercially synthesized and site-specifically bound to epoxy-derivatized glass slides in triplicate. Peptides were 10, 15, or 20 amino acids in length with an offset of either 2 or 3 amino acids. A total of 10 control and 45 peanut-allergic sera were assayed. Specific IgE and IgG4 were detected by using fluorochrome-labeled monoclonal secondary antibodies. Results: By using 15-mer and 20-mer peptides, we could define 11 antigenic regions, whereas only 5 were identifiable using 10-mers. Controls and patients produced IgG4 recognizing a comparable number of Ara h 2 peptides, although the dominant epitopes were distinct. As expected, patient IgE bound a larger number of Ara h 2 peptides (9.4% vs 0.9%). IgE and IgG4 epitopes recognized by patients were largely the same, and there was a positive association between IgE and IgG4 signal, suggesting coordinate regulation. Cluster analysis of peptide binding patterns confirmed the specificity of antibody-peptide interactions and was used to define 9 core epitopes ranging from 6 to 16 residues in length—7 of which (78%) agreed with previous mapping. Conclusion: Epitope mapping by microarray peptide immunoassay and cluster analysis reveals interpatient heterogeneity and a more detailed map. [Copyright &y& Elsevier]

Abstract: To assess the relative potency of the major peanut allergens, Ara h 1 and Ara h 2, we examined the relative ability of purified proteins to bind IgE on immunoblots, to cross-link allergen specific IgE in an in vitro assay of degranulation based on RBL SX-38 cells, and to bind IgE in the ImmunoCap® assay. Sera from 12 highly sensitive, peanut allergic patients were studied in all assays. IgE immunoblots with crude peanut extracts showed binding of IgE to multiple bands including the 63 kDa and 17–19 kDa bands that contain Ara h 1 and Ara h 2, respectively. In the functional assay, Ara h 2 was more potent than Ara h 1 in 11 of 12 sera tested with a median potency that was 52.5-fold more than Ara h 1 (P &#60; 0.005). Contrary to findings with the functional assay, IgE immunoblots with purified Ara h 1 and Ara h 2 showed substantially lighter binding of IgE to Ara h 2 compared with Ara h 1 (P = 0.02). The ImmunoCap® assay gave intermediate results with slightly more IgE binding to Ara h 2 than to Ara h 1 (P = 0.005). In conclusion, Ara h 2 is a very potent allergen and is much more potent than Ara h 1 for most sera using an in vitro assay of IgE cross-linking and cell activation. This finding is different from what was predicted based on immunoblots or with the ImmunoCap® assay. [Copyright &y& Elsevier]

The development of safe and effective immunotherapy for peanut allergy has been complicated by the high anaphylactic potential of native peanut extracts. We sought to map the T-cell epitopes of the major peanut allergen, Ara h 2 in order to develop T-cell targeted vaccines.A panel of eight peanut-specific CD4+ T-cell lines (TCL) was derived from eight peanut-allergic subjects and proliferative and cytokine responses to stimulation with a set of overlapping 20-mer peptides representing the entire sequence of Ara h 2 determined. Proliferation was assessed in 72 h assays via tritiated thymidine incorporation, while interleukin (IL)-5 and interferon (IFN)-γ production were assessed via sandwich enzyme-linked immunosorbent assay (ELISA) of cell culture supernatants.Eight of the 17 Ara h 2 peptides were recognized by one or more subjects, with the two peptides showing highest reactivity [Ara h 2 (19–38) and Ara h 2 (73–92)] being recognized by three subjects each. Adjoining peptides Ara h 2 (28–47) and Ara h 2 (100–119) induced proliferative responses in two subjects. Each of these peptides was associated with a Th2-type cytokine response.Two highly immunogenic T-cell reactive regions of Ara h 2 have been identified, Ara h 2 (19–47) and Ara h 2 (73–119), providing scope for the development of safe forms of immunotherapy for peanut allergy. [ABSTRACT FROM AUTHOR]

Peanuts are one of the allergens most often responsible for food allergy . Nevertheless, the occurrence of peanut allergy varies between countries and appears to result from both the amount of peanuts in the diet and the way they are consumed. In addition, processing influences the allergenicity of peanut proteins. The aim of the present study was to examine the effect of some thermal processes on the IgE binding capacity of peanut allergens, using sera of patients allergic to peanuts. Protein extracts were prepared from raw, roasted and boiled peanuts. In each case, the proteins were isolated, purified and characterised by electrophoresis. The immunoreactivity of each protein extract was analysed using the Enzyme Allergo Sorbent Test (EAST) and EAST inhibition with sera from 37 peanut allergic patients. Western blots were also performed. The biochemical analysis of the whole protein extracts revealed slight modifications in the protein composition, particularly after boiling. The IgE binding capacity of the boiled peanut protein extract was reduced by 50% compared to raw and roasted peanut extracts. This difference on the recognition of peanut allergens by patients’ IgE was confirmed by the inhibition assays. Impaired IgE recognition mainly involves proteins of low molecular weight. No difference in immunoreactivity was observed between raw and roasted peanut protein extracts. Roasting peanuts does not modify their allergenicity while boiling decreases it considerably. This decrease of boiled peanut allergenicity comes from a loss of some allergens into the boiling water. It may also be due to structural modifications of peanut proteins that occur in boiling water. [Copyright &y& Elsevier]

Background: Ara h 2 is a major peanut allergen recognized by IgE in more than 90% of patients. After electrophoretic separation the purified protein exists as a doublet, and sequences of one incomplete cDNA and one genomic clone for this allergen have been reported. Methods: Ara h 2 isoforms were purified and analyzed by mass spectroscopy, and PCR amplification products of Ara h 2 were cloned and sequenced. Results: Mass spectroscopy of purified Ara h 2 clearly identified a molecular doublet of 16,670 and 18,050 Daltons. Amplification of a peanut cDNA library using PCR primer pairs located at the amino- and carboxy-terminus revealed 2 bands separated by 50 base pairs, which we cloned and sequenced. Two types of complete cDNA clones were obtained, Ara h 2.01 and Ara h 2.02. Compared to Ara h 2.01 and the previously reported cDNA sequences, Ara h 2.02 is characterized by a 12 amino acid insertion starting at position 75 that contains a third repeat of the major IgE binding epitope DPYSPS. Conclusion: We demonstrated the molecular and genetic characteristics of two Ara h 2 isoforms, revealing that one, Ara h 2.02, might be the more potent allergen.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The maximum tolerated dose of peanut protein following peanut oral immunotherapy (POIT) is unknown because most research studies have not examined very high thresholds., Objective: To define the maximum dose tolerated by patients on POIT and severity of allergic reactions after a 1-month period of treatment discontinuation., Methods: In a phase 2 3-year POIT open-label study, we enrolled participants age 5 to 13 years with a 1-year build-up period followed by a 2-year daily maintenance dose of 3900 mg with assessment of the maximum tolerated dose using double-blind placebo-controlled food challenges (DBPCFCs) of 26,225 mg cumulative dose of peanut protein. The DBPCFC was performed at baseline, after 12-month build-up, at 2 year of maintenance, and after a 1-month period of treatment discontinuation. Biomarkers were assessed every 6 weeks for the first 6 months of therapy. A general linear mixed model was used for analysis., Results: The mean maximum cumulative tolerated dose after 12 months increased by 12,063 mg (P < .001) (n = 12), slightly decreased during maintenance (n = 11), and significantly decreased by 7593 mg after avoidance for 1 month (P = .03) (n = 6). Biomarker analysis revealed decreases in cytokine expression within the first 6 weeks of initiation of POIT and decreased peanut-IgG 4 and increased cytokine expression after 1 month of discontinuation. The DBPCFC reaction severity, examined through a symptom score with 1 point for each defined symptom, decreased after 12 months, but did not significantly change after 1 month of POIT discontinuation., Conclusions: The evaluation of POIT and sustained unresponsiveness by maximum tolerated dose by DBPCFCs in this small phase 2 trial showed that desensitization is diminished, with 100% loss of tolerated dose after 1 month of avoidance following 3 years of treatment., (Copyright © 2021. Published by Elsevier Inc.)

Peanut allergy is the leading pediatric food allergy. Many attempts have been made to reduce its allergenicity by processing. After roasting, Ara h 2 and its derivatives in the matrix were isolated by immunoaffinity chromatography (IAC). The structure and allergenicity of Ara h 2 were analyzed by circular dichroism, mass spectrometry (MS), western blotting, the enzyme-linked immunoassay, and cell modeling. Our results showed that a large portion of Ara h 2 was fragmented and cross-linked. Ara h 2 monomers accounted for only 13% of the total proteins after IAC purification. In addition, the structure of Ara h 2 changed after roasting. In addition to methylation and oxidation modification, the disulfide bonds of Ara h 2 were found to be rearranged after roasting. In the conformational structure of Ara h 2, the content of the α-helix decreased from 27.1 to 21.6% after roasting, while the content of the random coil increased from 29.1 to 34.3%. Six cleavage sites of trypsin were exposed, while three were covered. In terms of allergenicity, most of the cross-linking products were not recognized by patients' sera. Only one faint band around 40 kDa was observed in our blotting. For Ara h 2 monomers, roasting enhanced their IgE binding capacity and ability to stimulate the degranulation of basophils. The potential allergenicity increase of Ara h 2 monomers did not reflect the allergenicity change of Ara h 2 in the matrix due to the amount and property of its derivatives after roasting.

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSP OH S motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in Escherichia coli, Lactococcus lactis, Trichoplusia ni insect cell, and Chlamydomonas reinhardtii chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or E. coli -produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of Nicotiana benthamiana plants, using the plant virus-based magnICON ® transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in E. coli together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from N. benthamiana and E. coli were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils via IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the N. benthamiana -produced allergen was observed in comparison to the one induced by the E. coli product. Ara h 2 from N. benthamiana plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of N. benthamiana plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Üzülmez, Kalic, Mayr, Lengger, Tscheppe, Radauer, Hafner, Hemmer and Breiteneder.)