Serologic measurements for peanut allergy: Predicting clinical severity is complex.

Allergist-immunologists use serologic peanut allergy testing to maximize test sensitivity and specificity while minimizing cost and inconvenience. Recent advances toward this goal include a better understanding of specific IgE (sIgE) and component testing, epitope-sIgE assays, and basophil activation testing. Predicting reaction severity with serologic testing is challenged by a range of co-factors that influence reaction severity, such as the amount and form of any allergen consumed and comorbid disease. In 2020, the Allergy Immunology Joint Task Force on Practice Parameters recommended Ara h 2-sIgE as the most cost-effective diagnostic test for peanut allergy because of its superior performance, when compared with skin prick testing and serum IgE. Basophil activation testing, a functional test of allergic response not evaluated in the Joint Task Force on Practice Parameters guideline, is a promising option for both allergy diagnosis and prognosis. Similarly, epitope-sIgE testing may improve prediction of reaction thresholds, but further validation is needed. Despite advances in food allergy testing, many of these tools remain limited by cost, accessibility, and feasibility. In addition, there is a need for further research on how atopic dermatitis may be modifying serologic food allergy severity assessments. Given these limitations, allergy test selection requires a shared decision-making approach so that a patient's values and preferences regarding financial impact, inconvenience, and psychological effects are considered in the context of clinician expertise on the timing and use of optimized testing.

HLA alleles and sustained peanut consumption promote IgG4 responses in subjects protected from peanut allergy.

We investigated the interplay between genetics and oral peanut protein exposure in the determination of the immunological response to peanut using the targeted intervention in the LEAP clinical trial. We identified an association between peanut-specific IgG4 and HLA-DQA1*01:02 that was only observed in the presence of sustained oral peanut protein exposure. The association between IgG4 and HLA-DQA1*01:02 was driven by IgG4 specific for the Ara h 2 component. Once peanut consumption ceased, the association between IgG4-specific Ara h 2 and HLA-DQA1*01:02 was attenuated. The association was validated by observing expanded IgG4-specific epitopes in people who carried HLA-DQA1*01:02. Notably, we confirmed the previously reported associations with HLA-DQA1*01:02 and peanut allergy risk in the absence of oral peanut protein exposure. Interaction between HLA and presence or absence of exposure to peanut in an allergen- and epitope-specific manner implicates a mechanism of antigen recognition that is fundamental to driving immune responses related to allergy risk or protection.

Diagnostic accuracy of Ara h 2 for detecting peanut allergy in children.

BACKGROUND: Specific IgE to Ara h 2 is a diagnostic test for peanut allergy which may reduce the need for double-blind placebo-controlled food challenges (DBPCFC); however, guidance for using Ara h 2 in place of DBPCFCs has not been validated. OBJECTIVE: To prospectively evaluate 1) diagnostic accuracy of previously published Ara h 2 cut-off levels to diagnose peanut allergy in children and 2) costs. METHODS: A consecutive series of 150 children age 3.5 to 18 years was evaluated in secondary and tertiary settings in the Netherlands. sIgE to Ara h 2 was the index test, and oral peanut ingestion was the reference test. Oral peanut ingestion was home or supervised introduction for Ara h 2 ≤ 0.1, DBPCFC for 0.1-5.0 and open food challenge for ≥5.0. Costs were calculated using financial healthcare data. RESULTS: A conclusive reference test was performed in 113 children (75%). Sixty-four children (57%) had peanut allergy, as confirmed by a DBPCFC (27/47) or an open challenge (37/50). Forty-nine children (43%) were considered peanut-tolerant after peanut introduction (19/19), a DBPCFC (20/47) or an open challenge (10/50). Area under the curve for Ara h 2 was 0.94 (95% CI 0.90-0.98). The diagnostic flow chart correctly classified 26/26 (100%; 84-100) of children with Ara h 2 ≤ 0.1 as peanut-tolerant and 34/35 (97%; 83-100) of children with Ara h 2 ≥ 5.0 as peanut-allergic. At a cut-off of ≤0.1 and ≥5.0, a sensitivity of respectively 100% (93-100) and 53% (38-67) was observed and a specificity of 53% (38-67) and 98% (87-100). Mean annual costs of the flow chart were estimated as €320-€636 per patient lower than following national allergy guidelines. CONCLUSIONS: In this diagnostic accuracy study, which did not take into account pretest probability, we have validated previously published Ara h 2 cut-off levels which are associated with peanut tolerance and allergy.

Sensitization Potency of Sunflower Seed Protein in a Mouse Model: Identification of 2S-Albumins More Allergenic Than SFA-8.

SCOPE: Food allergy to sunflower seed (SFS) protein is not frequent and only non-specific lipid transfert protein (nsLTP) Hel a 3 is officially recognized as a food allergen. Out of the eleven seed storage 2S-albumins (SESA) detected in SFS, only SFA-8 allergenicity has been investigated so far. The study aimed then to evaluate SFS protein allergenicity and particularly, to compare the sensitization potency of SESA in a mouse model. METHODS AND RESULTS: The most abundant SESA and nsLTP were isolated from SFS through a combination of chromatographic methods. Purified proteins were then used to measure specific IgG1 and IgE responses in BALB/c mice orally sensitized to different SFS protein isolates. The study, thus, confirmed the allergenicity of SFA-8 and Hel a 3 but mice were also highly sensitized to other SESA such as SESA2-1 or SESA20-2. Furthermore, competitive inhibition of IgE-binding revealed that SFA-8 IgE-reactivity was due to cross-reactivity with other SESA. 11S-globulins were weakly immunogenic and were rapidly degraded in an in vitro model of gastroduodenal digestion. In contrast, Hel a 3, SESA2-1 and SFA-8 were more resistant to proteolysis and gastroduodenal digestion did not affect their IgE-reactivity. CONCLUSIONS: SESA2-1 or SESA20-2 were more potent allergens than SFA-8 in this mouse model. Allergenicity of SESA must be now confirmed in SFS-allergic patients.

The impact of a baked muffin matrix on the bioaccessibility and IgE reactivity of egg and peanut allergens.

Baked matrices, such as muffin, may help to promote tolerance to food allergens by modifying allergen structure, digestibility, and capacity to stimulate the immune responses. However, the impact of the muffin matrix on the bioaccessibility of allergens in the gastrointestinal tract is not well understood. Muffin containing egg and peanut was subjected to in vitro oral-gastro-duodenal digestion. During gastric digestion, the majority of the egg allergen Gal d 2 and the peanut allergens Ara h 1 and 3 were not bioaccessible. Subsequent duodenal digestion increased allergen bioaccessibility with Gal d 2 and the peanut allergen Ara h 2 proving highly resistant to digestion. The IgE reactivity of bioaccessible peanut allergens was retained to a greater extent than that of egg allergens after oral-gastric digestion. The starch and gluten-rich muffin matrix modifies allergen bioaccessiblity in a manner more similar to baked matrices such as bread, than low water activity matrices such as cookies.

Predicting the allergenicity of legume proteins using a PBMC gene expression assay.

BACKGROUND: Food proteins differ in their allergenic potential. Currently, there is no predictive and validated bio-assay to evaluate the allergenicity of novel food proteins. The objective of this study was to investigate the potential of a human peripheral blood mononuclear cell (PBMC) gene expression assay to identify biomarkers to predict the allergenicity of legume proteins. RESULTS: PBMCs from healthy donors were exposed to weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine) in three experiments. Possible biomarkers for allergenicity were investigated by exposing PBMCs to a protein pair of weakly (white bean) and strongly allergenic (soybean) 7S globulins in a pilot experiment. Gene expression was measured by RNA-sequencing and differentially expressed genes were selected as biomarkers. 153 genes were identified as having significantly different expression levels to the 7S globulin of white bean compared to soybean. Inclusion of multiple protein pairs from 2S albumins (lupine and peanut) and 7S globulins (white bean and soybean) in a larger study, led to the selection of CCL2, CCL7, and RASD2 as biomarkers to distinguish weakly from strongly allergenic proteins. The relevance of these three biomarkers was confirmed by qPCR when PBMCs were exposed to a larger panel of weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine). CONCLUSIONS: The PBMC gene expression assay can potentially distinguish weakly from strongly allergenic legume proteins within a protein family, though it will be challenging to develop a generic method for all protein families from plant and animal sources. Graded responses within a protein family might be of more value in allergenicity prediction instead of a yes or no classification.

Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1.

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.

Peanuts in the air - clinical and experimental studies.

BACKGROUND: Allergic reactions to food allergens usually occur after ingestion. However, fear of reactions to airborne peanut is a common concern for people with peanut allergy. There are no scientific reports on severe reactions with airborne peanut allergen. OBJECTIVE: To investigate the occurrence of allergic reactions in peanut-allergic children undergoing airborne peanut challenge and to determine levels of airborne peanut protein in a separate experimental evaluation. METHODS: Eighty-four children with peanut allergy underwent an airborne peanut challenge, 0.5 m from a bowl of peanuts for 30 min under controlled conditions. In a separate experiment, airborne peanut proteins from roasted and dry-roasted peanuts were collected at varying distances and at varying times with an electret SensAbues filter connected to an air pump. Collected airborne peanut proteins were extracted, dissolved and detected by ELISA. Basophil activation test was used to confirm biological activity. RESULTS: No moderate/severe allergic reactions to airborne peanut allergens were observed. Two children (2%) had mild rhino-conjunctivitis which required no treatment. The IgE-antibodies to peanut or Ara h 2 did not predict a reaction. In the experimental set-up, biological active peanut proteins were detected, in a very low amount, in median 166 ng/ml for dry-roasted and 33 ng/ml for roasted peanuts and decreased dramatically when the collection occurred at a greater distance (0.5-2 m) from the peanut source. Increased exposure time did affect the amount of collected peanut protein at 0 m, and the highest median was obtained after 60 min (p = .012); for time trend p = .0006. CONCLUSIONS AND CLINICAL RELEVANCE: Allergic reactions to airborne peanut proteins are rare and cannot be predicted by high levels of IgE-antibodies to peanut or Ara h 2. Only small amounts of biologically active peanut proteins were detected in the air and seem unlikely to trigger moderate/severe allergic reactions.

Ara h 2-specific IgE is superior to whole peanut extract-based serology or skin prick test for diagnosis of peanut allergy in infancy.

BACKGROUND: Screening of high-risk infants for peanut allergy (PA) before introduction is now recommended in the United States, but the optimal approach is not clear. OBJECTIVE: We sought to compare the diagnostic test characteristics of peanut skin prick test (SPT), peanut-specific IgE (sIgE), and sIgE to peanut components in a screening population of infants before known peanut exposure. METHODS: Infants aged 4 to 11 months with (1) no history of peanut ingestion, testing, or reaction and (2) (a) moderate-severe eczema, (b) history of food allergy, and/or (c) first-degree relative with a history of PA received peanut SPT, peanut-sIgE and component-IgE testing, and, depending on SPT wheal size, oral food challenge or observed feeding. Receiver-operator characteristic areas under the curve (AUCs) were compared, and diagnostic sensitivity and specificity were calculated. RESULTS: A total of 321 subjects completed the enrollment visit (median age, 7.2 months; 58% males), and 37 (11%) were found to have PA. Overall, Ara h 2-sIgE at a cutoff point of 0.1 kUa/L discriminated between allergic and nonallergic best (AUC, 0.96; sensitivity, 94%; specificity, 98%), compared with peanut-sIgE at 0.1 kUa/L (AUC, 0.89; sensitivity, 100%; specificity, 78%) or 0.35 kUa/L (AUC, 0.91; sensitivity, 97%; specificity, 86%), or SPT at wheal size 3 mm (AUC, 0.90; sensitivity, 92%; specificity, 88%) or 8 mm (AUC, 0.87; sensitivity, 73%; specificity, 99%). Ara h 1-sIgE and Ara h 3-sIgE did not add to prediction of PA when included in a model with Ara h 2-sIgE, and Ara h 8-sIgE discriminated poorly (AUC, 0.51). CONCLUSIONS: Measurement of only Ara h 2-sIgE should be considered if screening of high-risk infants is performed before peanut introduction.

Epicutaneous immunotherapy for treatment of peanut allergy: Follow-up from the Consortium for Food Allergy Research.

BACKGROUND: Consortium for Food Allergy Research investigators previously reported 52-week outcomes from a randomized controlled trial of peanut epicutaneous immunotherapy, observing modest and statistically significant induction of desensitization, highest in children ages 4 to 11 years. OBJECTIVE: We sought to evaluate changes in efficacy, safety, and mechanistic parameters following extended open-label peanut epicutaneous immunotherapy. METHODS: Peanut-allergic participants (4-25 years) received 52 weeks of placebo (PLB), Viaskin Peanut 100 µg (VP100) or 250 µg (VP250), and then crossed over to VP250 for PLB (PLB-VP250) and VP100 (VP100-VP250) participants and continued treatment for VP250 participants (total = 130 weeks of active epicutaneous immunotherapy). Efficacy was assessed by double-blind, placebo-controlled food challenge (5044 mg peanut protein), and adherence, safety, and mechanistic parameters were evaluated. RESULTS: At week 130, desensitization success was achieved in 1 of 20 (5%) PLB-VP250, 5 of 24 (20.8%) VP100-VP250, and 9 of 25 (36%) VP250 participants, with median successfully consumed dose change from baseline of 11.5 mg, 141.5 mg, and 400 mg, respectively. Median age (years) for week 130 desensitization success was 6.2 years (interquartile range, 5.2-9.1) versus 9.4 years (interquartile range, 7.6-12.8) for failures (P < .001). Adherence was 96%. Adverse reactions were predominantly local patch-site reactions. Significant increases in peanut- and Ara h2-specific IgG4 observed at week 52 persisted to week 130. By a post hoc analysis, there were no statistically significant increases from week 52 to week 130 in either desensitization success or successfully consumed dose. CONCLUSIONS: Extended treatment with VP250 was well tolerated, and desensitization observed at week 52 persisted between weeks 52 and 130. Treatment success was observed predominantly in younger participants, with younger age at initiation of active therapy an important predictor of success.

Epitope mapping of the major allergen 2S albumin from pine nut.

The epitopes of the major allergen of pine nut, Pin p 1, were analyzed using a peptide library and sera from patients with clinical allergy to pine nut in order to deepen into the allergenic characteristics of Pin p 1. Analyses of epitope similarities and epitopes location in a 3D-model were also performed. Results showed that three main regions of Pin p 1 containing 5 epitopes were recognized by patient sera IgE. The epitopes of Pin p 1 had important similarities with epitopes of allergenic 2S albumins from peanut (Ara h 2 and 6) and Brazil nut (Ber e 1). The epitopes of Pin p 1 were found in α-helices and coils in the 3D protein structure. Interestingly, all epitopes were found to be well-exposed in the protein surface, which suggests facile access for IgE-binding to the structure of Pin p 1 which is known to be highly resistant.

The importance of the 2S albumins for allergenicity and cross-reactivity of peanuts, tree nuts, and sesame seeds.

Allergies to peanuts, tree nuts, and sesame seeds are among the most important food-related causes of anaphylaxis. Important clinical questions include: Why is there a variable occurrence of coallergy among these foods and Is this immunologically mediated? The clinical and immunologic data summarized here suggest an immunologic basis for these coallergies that is based on similarities among the 2S albumins. Data from component resolved diagnostics have highlighted the relationship between IgE binding to these allergens and the presence of IgE-mediated food allergy. Furthermore, in vitro and in vivo experiments provide strong evidence that the 2S albumins are the most important allergens in peanuts for inducing an allergic effector response. Although the 2S albumins are diverse, they have a common disulfide-linked core with similar physicochemical properties that make them prime candidates to explain much of the observed coallergy among peanuts, tree nuts, and sesame seeds. The well-established frequency of cashew and pistachio nut coallergy (64%-100%) highlights how the structural similarities among their 2S albumins may account for observed clinical cross-reactivity. A complete understanding of the physicochemical properties of the 2S albumins in peanuts, tree nuts, and sesame seeds will enhance our ability to diagnose, treat, and ultimately prevent these allergies.

Component resolved diagnostics in peanut sensitized children with and without a history of clinical reaction.

Background: Serum Peanut-specific-IgE (PN-sIgE) and peanut-component-resolved-diagnostics (CRD) are often ordered simultaneously in the evaluation for peanut allergy. Results often guide the plans for peanut oral challenge. However, the clinical utility of CRD at different total PN-sIgE levels is unclear. A commonly used predefined CRD Ara h2 cutoff value in the literature predicting probability of peanut challenge outcomes is 0.35kUA/L. Objective: To examine the utility of CRD in patients with and without a history of clinical reactivity to peanut (PN). Methods: This was a retrospective chart review of 196 children with PN-sIgE and CRD testing, of which, 98 patients had a clinical history of an IgE-mediated reaction when exposed to PN and 98 did not. The Fisher's exact test was used to assess the relationship between CRD and PN-sIgE at different cutoff levels, McNemar test and Gwet's approach (AC1 statistic) were used to examine agreement between CRD and PN-sIgE, and logistic regression was used to assess differences in the findings between patients with and without reaction history. Results: Ara h 1, 2, 3, or 9 (ARAH) levels ≤0.35 kUA/L were significantly associated with PN-sIgE levels <2 kUA/L rather than ≥2 kUA/L (p < 0.0001). When the ARAH threshold was increased to 1 kUA/L and 2 kUA/L, these thresholds were still significantly associated with PN-sIgE levels of <2, <5, and <14 kUA/L. These findings were not significantly different in patients with and without a history of clinical reactivity. Conclusion: ARAH values correlated with PN-sIgE. Regardless of clinical history, ARAH levels are unlikely to be below 0.35, 1, or 2 kUA/L if the PN-sIgE level is >2 kUA/L. Thus, if possible, practitioners should consider PN-sIgE rather than automatically ordering CRD with PN-sIgE every time. Laboratory procedures that allow automatically and reflexively adding CRD when the PN-sIgE level is ≤5 kUA/L can be helpful. However, further studies are needed in subjects with challenge-proven PN allergy.

Transcriptional frameshifts contribute to protein allergenicity.

Transcription infidelity (TI) is a mechanism that increases RNA and protein diversity. We found that single-base omissions (i.e., gaps) occurred at significantly higher rates in the RNA of highly allergenic legumes. Transcripts from peanut, soybean, sesame, and mite allergens contained a higher density of gaps than those of nonallergens. Allergen transcripts translate into proteins with a cationic carboxy terminus depleted in hydrophobic residues. In mice, recombinant TI variants of the peanut allergen Ara h 2, but not the canonical allergen itself, induced, without adjuvant, the production of anaphylactogenic specific IgE (sIgE), binding to linear epitopes on both canonical and TI segments of the TI variants. The removal of cationic proteins from bovine lactoserum markedly reduced its capacity to induce sIgE. In peanut-allergic children, the sIgE reactivity was directed toward both canonical and TI segments of Ara h 2 variants. We discovered 2 peanut allergens, which we believe to be previously unreported, because of their RNA-DNA divergence gap patterns and TI peptide amino acid composition. Finally, we showed that the sIgE of children with IgE-negative milk allergy targeted cationic proteins in lactoserum. We propose that it is not the canonical allergens, but their TI variants, that initiate sIgE isotype switching, while both canonical and TI variants elicit clinical allergic reactions.

Effect of heat treatment on the conformational stability of intact and cleaved forms ofthe peanut allergen Ara h 6 in relation to its IgE-binding potency.

This work reports on theeffect of heat treatment on the protein conformational stabilityof intact and post-translationallycleaved peanut allergen Ara h 6 in relation to IgE-binding. Intact and post-translationallycleaved Ara h 6 are structurally similar and theirstrong resistance to denaturant-inducedunfolding is comparable. Only upon exposure toautoclave conditions the twoforms of Ara h 6 demonstrated susceptibility toirreversible denaturationresulting in a significant decrease in IgE-binding potency. Thisreduction isfor the intact protein more pronounced than for than for the cleaved form. This isattributed to less conformational constrains of the cleaved form comparedtointact, as suggested by the 2-fold lower activation energy for unfoldingfound for the cleavedform. Overall, harsh conditionsare required to denature Ara h 6 and to significantly reduce its IgE-bindingpotency. The cleavedform possesses more resistance to such denaturation than the intactform.

The Effect of the Food Matrix on the In Vitro Bio-Accessibility and IgE Reactivity of Peanut Allergens.

SCOPE: Factors such as food processing, the food matrix, and antacid medication may affect the bio-accessibility of proteins in the gastrointestinal tract and hence their allergenic activity. However, at present they are poorly understood. METHODS AND RESULTS: Roasted peanut flour was incorporated into either a chocolate dessert or cookie matrix and bio-accessibility were assessed using an in vitro digestion system comprising a model chew and simulated gastric and duodenal digestion. Protein digestion was monitored by SDS-PAGE and immunoreactivity analyzed by immunoblotting and immunoassay. IgE reactivity was assessed by immunoassay using serum panels from peanut-allergic subjects. Roasted peanut flour proteins proved highly digestible following gastro-duodenal digestion even when incurred into a food matrix, with only low molecular weight polypeptides of Mr < 8 kDa remaining. When gastric digestion was performed at pH 6.5 (simulating the effect of antacid medication), peanut proteins are not digested; subsequent duodenal digestion is also limited. IgE reactivity of the major peanut allergens Ara h 1, Ara h 2, and Ara h 6, although reduced, was retained after oral-gastro-duodenal digestion irrespective of digestion conditions employed. CONCLUSION: Peanut allergen bio-accessibility is unaffected by the dessert or cookie matrices whilst high intra-gastric pH conditions render allergens more resistant to digestion.

Ara h 2 is the dominant peanut allergen despite similarities with Ara h 6.

BACKGROUND: Arachis hypogaea 2 (Ara h 2)-specific IgE is to date the best serologic marker to diagnose peanut allergy. Ara h 6 shares approximately 60% sequence identity and multiple epitopes with Ara h 2. OBJECTIVE: Our aim was to assess the diagnostic utility and relative importance of Ara h 2 and Ara h 6 in peanut allergy. METHODS: A cohort 100 of children was studied. The cohort included chidren who had peanut allergy, children who were sensitized to but tolerant of peanut, and children who were neither sensitized nor allergic to peanut. Levels of specific IgE to peanut and individual allergens were quantified by using ImmunoCAP. ImmunoCAP inhibition experiments and mast cell activation tests in response to both Ara h 2 and Ara h 6 were performed. Statistical analyses were done using SPSS version 14 and Prism version 7 software. RESULTS: Ara h 2-specific IgE and Ara h 6-specific IgE showed the greatest diagnostic accuracy for peanut allergy when compared with specific IgE to peanut and other peanut allergens. Most patients with peanut allergy were sensitized to both Ara h 2 and Ara h 6. Ara h 2 reduced Ara h 2-specific IgE binding more than Ara h 6 did (P < .001), whereas Ara h 6-specific IgE binding was inhibited to a similar degree by Ara h 2 and Ara h 6 (P = .432). In the mast cell activation test, Ara h 2 induced significantly greater maximal reactivity (P = .001) and a lower half maximal effective concentration (P = .002) than did Ara h 6 when testing cosensitized individuals. CONCLUSIONS: Ara h 2-specific IgE and Ara h 6-specific IgE provide the greatest accuracy to diagnose peanut allergy. Ara h 2 is the dominant conglutin in peanut allergy in the United Kingdom, despite a degree of cross-reactivity with Ara h 6.

A Single Monoclonal Antibody against the Peanut Allergen Ara h 2 Protects against Systemic and Local Peanut Allergy.

BACKGROUND: Peanut allergy is the most prevalent and dangerous food allergy. Peanuts consist of a large number of different allergens and peanut-allergic patients are frequently sensitized to multiple allergens. Hence, conventional desensitization approaches aim at targeting as many allergens as possible. METHODS: The monoclonal anti-Ara h 2 antibody (mAb) was produced by hybridoma cells derived from WT BALB/c mice after immunization with a vaccine based on virus-like particles coupled to Ara h 2. BALB/c mice were sensitized intraperitoneally with peanut extract absorbed to alum and mAbs were applied i.v. Challenge was performed the next day with the whole peanut extract intravenously and via skin prick test. RESULTS: Here we show in peanut-allergic mice that a single high-affinity mAb specific for Ara h 2 is able to block systemic and local allergic reactions induced by the complex peanut extract. We confirm in vitro binding of the mAb to the inhibitory low-affinity FcγRIIb receptor using a sensitive biosensor and demonstrate in vivo that protection was dependent on FcγRIIb. CONCLUSION: A single mAb specific for Ara h 2 is able to improve local and systemic allergic symptoms induced by the whole allergen mixture.

Origins and clonal convergence of gastrointestinal IgE+ B cells in human peanut allergy.

B cells in human food allergy have been studied predominantly in the blood. Little is known about IgE+ B cells or plasma cells in tissues exposed to dietary antigens. We characterized IgE+ clones in blood, stomach, duodenum, and esophagus of 19 peanut-allergic patients, using high-throughput DNA sequencing. IgE+ cells in allergic patients are enriched in stomach and duodenum, and have a plasma cell phenotype. Clonally related IgE+ and non-IgE-expressing cell frequencies in tissues suggest local isotype switching, including transitions between IgA and IgE isotypes. Highly similar antibody sequences specific for peanut allergen Ara h 2 are shared between patients, indicating that common immunoglobulin genetic rearrangements may contribute to pathogenesis. These data define the gastrointestinal tract as a reservoir of IgE+ B lineage cells in food allergy.