Peanut protein acts as a TH2 adjuvant by inducing RALDH2 in human antigen-presenting cells.

BACKGROUND: Peanut is a potent inducer of proallergenic TH2 responses in susceptible individuals. Antigen-presenting cells (APCs) including dendritic cells and monocytes instruct naive T cells to differentiate into various effector cells, determining immune responses such as allergy and tolerance. OBJECTIVE: We sought to detect peanut protein (PN)-induced changes in gene expression in human myeloid dendritic cells (mDCs) and monocytes, identify signaling receptors that mediate these changes, and assess how PN-induced genes in mDCs impact their ability to promote T-cell differentiation. METHODS: mDCs, monocytes, and naive CD4+ T cells were isolated from blood bank donors and peanut-allergic patients. APCs were incubated with PN and other stimulants, and gene expression was measured using microarray and RT quantitative PCR. To assess T-cell differentiation, mDCs were cocultured with naive TH cells. RESULTS: PN induced a unique gene expression profile in mDCs, including the gene that encodes retinaldehyde dehydrogenase 2 (RALDH2), a rate-limiting enzyme in the retinoic acid (RA)-producing pathway. Stimulation of mDCs with PN also induced a 7-fold increase in the enzymatic activity of RALDH2. Blocking antibodies against Toll-like receptor (TLR)1/TLR2, as well as small interfering RNA targeting TLR1/TLR2, reduced the expression of RALDH2 in PN-stimulated APCs by 70%. Naive TH cells cocultured with PN-stimulated mDCs showed an RA-dependent 4-fold increase in production of IL-5 and expression of integrin α4ß7. CONCLUSIONS: PN induces RALDH2 in human APCs by signaling through the TLR1/TLR2 heterodimer. This leads to production of RA, which acts on TH cells to induce IL-5 and gut-homing integrin. RALDH2 induction by PN in APCs and RA-promoted TH2 differentiation could be an important factor determining allergic responses to peanut.

IgE to epitopes of Ara h 2 enhance the diagnostic accuracy of Ara h 2-specific IgE.

BACKGROUND: Understanding the discrepancy between IgE sensitization and allergic reactions to peanut could facilitate diagnosis and lead to novel means of treating peanut allergy. OBJECTIVE: To identify differences in IgE and IgG4 binding to peanut peptides between peanut-allergic (PA) and peanut-sensitized but tolerant (PS) children. METHODS: PA (n = 56), PS (n = 42) and nonsensitized nonallergic (NA, n = 10) patients were studied. Synthetic overlapping 15-mer peptides of peanut allergens (Ara h 1-11) were spotted onto microarray slides, and patients' samples were tested for IgE and IgG4 binding using immunofluorescence. IgE and IgG4 levels to selected peptides were quantified using ImmunoCAP. Diagnostic model comparisons were performed using likelihood-ratio tests between each specified nominal logistic regression models. RESULTS: Seven peptides on Ara h 1, Ara h 2, and Ara h 3 were bound more by IgE of PA compared to PS patients on the microarray. IgE binding to one peptide on Ara h 5 and IgG4 binding to one Ara h 9 peptide were greater in PS than in PA patients. Using ImmunoCAP, IgE to the Ara h 2 peptides enhanced the diagnostic accuracy of Ara h 2-specific IgE. Ratios of IgG4/IgE to 4 out of the 7 peptides were higher in PS than in PA subjects. CONCLUSIONS: Ara h 2 peptide-specific IgE added diagnostic value to Ara h 2-specific IgE. Ability of peptide-specific IgG4 to surmount their IgE counterpart seems to be important in established peanut tolerance.

Peanut Allergy: Characteristics and Approaches for Mitigation.

Peanut allergy has garnered significant attention because of the high sensitization rate, increase in allergy, and severity of the reaction. Sufficiently reliable therapies and efficient mitigating techniques to combat peanut allergy are still lacking. Current management relies on avoiding peanuts and nuts and seeds with homologous proteins, although adverse events mostly occur with accidental ingestion. There is a need for hypoallergenic peanut products to protect sensitized individuals and perhaps serve as immunotherapeutic products. Alongside traditional practices of thermal and chemical treatment, novel processing approaches such as high-pressure processing, pulsed ultraviolet light, high-intensity ultrasound, irradiation, and pulsed electric field have been performed toward reducing the immunoreactivity of peanut. Covalent and noncovalent chemical modifications to proteins also have the tendency to alter peanut allergenicity. Enzymatic hydrolysis seems to be the most advantageous technique in diminishing the allergenic potential of peanut. Furthermore, the combined processing approach (hurdle technologies) such as enzymatic hydrolysis followed by, or in conjunction with, roasting, high pressure and heat, ultrasound with enzymatic treatment, or germination have shown a significant reduction of peanut immunoreactivity and may emerge as useful techniques in reducing the allergenicity of peanut and other foods. This study represents our current knowledge about the alterations in allergenic properties of peanut via different processing mechanisms as well as evaluating its future potential, geographical based data on increasing sensitization, clinical relevance, eliciting dose, and current management of peanut allergy. Furthermore, the molecular characteristics and clinical relevance of peanut allergens have been discussed.

Differences in the Uptake of Ara h 3 from Raw and Roasted Peanut by Monocyte-Derived Dendritic Cells.

Roasting has been implicated in the increase of peanut allergenicity due to the chemical reactions that occur during the process. However, this increase is not fully understood, and little information is available regarding the role of roasted peanut allergens in the initial phase of allergy, where dendritic cells (DCs) play a key role. We sought to analyze differences in the internalization of Ara h 3 from raw and roasted peanut by immature monocyte-derived DCs (MDDCs) and the implication of the mannose receptor in the uptake. Ara h 3 was purified from raw and roasted peanut (Ara h 3-raw and Ara h 3-roas) and labeled with a fluorescent dye. The labeled allergens were added to MDDCs obtained from 7 donors and internalization was analyzed after 10, 30, and 120 min by flow cytometry. In parallel, mannan, which blocks the mannose receptor, was added 30 min before adding the labeled allergens. Results showed that the internalization of Ara h 3-roas by MDDCs was significantly increased at every time point. However, the increase in the internalization of Ara h 3-raw was only significant after 2 h of incubation. Ara h 3-roas had an enhanced capacity to be internalized by MDDCs in comparison with Ara h 3-raw at every time point. Blocking the mannose receptor decreased the internalization of Ara h 3-roas but not Ara h 3-raw. In conclusion, the internalization of Ara h 3-roas by the MDDCs is enhanced when compared to Ara h 3-raw, and the mannose receptor might be implicated in this enhancement.

Immunotherapy using algal-produced Ara h 1 core domain suppresses peanut allergy in mice.

Peanut allergy is an IgE-mediated adverse reaction to a subset of proteins found in peanuts. Immunotherapy aims to desensitize allergic patients through repeated and escalating exposures for several months to years using extracts or flours. The complex mix of proteins and variability between preparations complicates immunotherapy studies. Moreover, peanut immunotherapy is associated with frequent negative side effects and patients are often at risk of allergic reactions once immunotherapy is discontinued. Allergen-specific approaches using recombinant proteins are an attractive alternative because they allow more precise dosing and the opportunity to engineer proteins with improved safety profiles. We tested whether Ara h 1 and Ara h 2, two major peanut allergens, could be produced using chloroplast of the unicellular eukaryotic alga, Chlamydomonas reinhardtii. C. reinhardtii is novel host for producing allergens that is genetically tractable, inexpensive and easy to grow, and is able to produce more complex proteins than bacterial hosts. Compared to the native proteins, algal-produced Ara h 1 core domain and Ara h 2 have a reduced affinity for IgE from peanut-allergic patients. We further found that immunotherapy using algal-produced Ara h 1 core domain confers protection from peanut-induced anaphylaxis in a murine model of peanut allergy.

Structure and function of the peanut panallergen Ara h 8.

The incidence of peanut allergy continues to rise in the United States and Europe. Whereas exposure to the major allergens Ara h 1, 2, 3, and 6 can cause fatal anaphylaxis, exposure to the minor allergens usually does not. Ara h 8 is a minor allergen. Importantly, it is the minor food allergens that are thought to be responsible for oral allergy syndrome (OAS), in which sensitization to airborne allergens causes a Type 2 allergic reaction to ingested foods. Furthermore, it is believed that similar protein structure rather than a similar linear sequence is the cause of OAS. Bet v 1 from birch pollen is a common sensitizing agent, and OAS results when patients consume certain fruits, vegetables, tree nuts, and peanuts. Here, we report the three-dimensional structure of Ara h 8, a Bet v 1 homolog. The overall fold is very similar to that of Bet v 1, Api g 1 (celery), Gly m 4 (soy), and Pru av 1 (cherry). Ara h 8 binds the isoflavones quercetin and apigenin as well as resveratrol avidly.

The molecular basis of peanut allergy.

Peanut allergens can trigger a potent and sometimes dangerous immune response in an increasing number of people. The molecular structures of these allergens form the basis for understanding this response. This review describes the currently known peanut allergen structures and discusses how modifications both enzymatic and non-enzymatic affect digestion, innate immune recognition, and IgE interactions. The allergen structures help explain cross-reactivity among allergens from different sources, which is useful in improving patient diagnostics. Surprisingly, it was recently noted that similar short peptide sequences among unrelated peanut allergens could also be a source of cross-reactivity. The molecular features of peanut allergens continue to inform predictions and provide new research directions in the study of allergic disease.

IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3.

BACKGROUND: Ara h 1, a vicilin; Ara h 2, a 2S albumin; and Ara h 3, a legumin, are major peanut allergens. Ara h 2 is an important predictor of clinical reactivity to peanut, but cosensitization to all 3 allergens is correlated with the severity of patients' symptoms. OBJECTIVE: We investigated whether cosensitization to these 3 allergens is caused by IgE cross-reactivity, despite the fact that they do not display obvious structural or sequence similarities. METHODS: IgE cross-inhibitions were performed with purified Ara h 1, Ara h 2, and Ara h 3 and IgG-depleted sera from 10 patients with peanut allergy. After an in silico search for similar peptides, IgE ELISA inhibition assays with synthetic peptides were performed. RESULTS: Ara h 2 inhibited IgE binding to Ara h 1 (average, 86% ± 13%) and Ara h 3 (average, 96% ± 6%). IgE binding to Ara h 2 was inhibited by Ara h 1 by 78% ± 15% and by Ara h 3 by 80% ± 6%. A subsequent sequence comparison showed that these nonhomologous allergens contained several similar surface-exposed peptides. IgE binding to Ara h 2-derived peptides was completely inhibited by Ara h 1 and Ara h 3. A mixture of these peptides reduced IgE binding to Ara h 1 and Ara h 3 by 20% to 60% and to Ara h 2 by 49% to 89%. CONCLUSION: Occurrence of similar sequences in the 3 major peanut allergens accounts for the high extent of cross-reactivity among them.

Distribution of peanut protein in the home environment.

BACKGROUND: To halt the increase in peanut allergy, we must determine how children become sensitized to peanut. High household peanut consumption used as an indirect marker of environmental peanut exposure is associated with the development of peanut allergy. OBJECTIVE: We sought to validate a method to quantify environmental peanut exposure, to determine how peanut is transferred into the environment after peanut consumption, and to determine whether environmental peanut persists despite cleaning. METHODS: After initial comparative studies among 3 ELISA kits, we validated and used the Veratox polyclonal peanut ELISA to assess peanut protein concentrations in dust and air and on household surfaces, bedding, furnishings, hand wipes, and saliva. RESULTS: The Veratox polyclonal peanut ELISA had the best rate of recovery of an independent peanut standard. We demonstrated 100% sensitivity and specificity and a less than 15% coefficient of variation for intra-assay, interassay, and interoperator variability. There was high within-home correlation for peanut protein levels in dust and household surface wipes. Airborne peanut levels were lower than the limit of quantitation for the Veratox polyclonal peanut ELISA in a number of simulated scenarios, except for a brief period directly above peanuts being deshelled. Peanut protein persisted on hands and in saliva 3 hours after peanut consumption. Peanut protein was completely removed from granite tables after cleaning with detergent, and levels were reduced but still present after detergent cleaning of laminate and wooden table surfaces, pillows, and sofa covers. CONCLUSIONS: Peanut spread easily around the home and might be resistant to usual cleaning methods. Peanut protein can be transferred into the environment by means of hand transfer and saliva but is unlikely to be aerosolized.

Structure and IgE Cross-Reactivity among Cashew, Pistachio, Walnut, and Peanut Vicilin-Buried Peptides.

Peanut and tree-nut allergies are frequently comorbid for reasons not completely understood. Vicilin-buried peptides (VBPs) are an emerging family of food allergens whose conserved structural fold could mediate peanut/tree-nut co-allergy. Peptide microarrays were used to identify immunoglobulin E (IgE) epitopes from the N-terminus of the vicilin allergens Ara h 1, Ana o 1, Jug r 2, and Pis v 3 using serum from three patient diagnosis groups: monoallergic to either peanuts or cashew/pistachio, or dual allergic. IgE binding peptides were highly prevalent in the VBP domains AH1.1, AO1.1, JR2.1, and PV3.1, but not in AO1.2, JR2.2, JR2.3, and PV3.2 nor the unstructured regions. The IgE profiles did not correlate with diagnosis group. The structure of the VBPs from cashew and pistachio was solved using solution-NMR. Comparisons of structural features suggest that the VBP scaffold from peanuts and tree-nuts can support cross-reactivity. This may help understand comorbidity and cross-reactivity despite a distant evolutionary origin.

IgE and IgG4 epitopes of the peanut allergens shift following oral immunotherapy.

Background: Oral immunotherapy (OIT) with peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Aimmune Therapeutics) is an FDA-approved treatment to desensitize peanut allergic participants. Objective: Here we assessed shifts in IgE and IgG4 binding to peanut allergens and their epitopes recognized by United States (US) peanut allergic participants (n = 20) enrolled in phase 3 PTAH OIT clinical trials. Methods: Pre- and post- trial participant sera were collected approximately 12 months apart and tested for IgE binding to intact peanut proteins via ImmunoCAP ISAC immunoassays. IgE and IgG4 linear epitopes were identified based on binding to synthetic overlapping 15-mer linear peptides of 10 peanut allergens (Ara h 1-11) synthesized on microarray slides. Results: Statistically significant decreases in IgE binding were identified for intact Ara h 2, 3, and 6, and known and newly identified IgE epitopes were shown to exhibit shifts towards IgG4 binding post-OIT, with most linear peptides having increased IgG4 binding after treatment with PTAH. While PTAH does not seem to alter the actual peptide binding patterns significantly after one year of treatment, the IgE and IgG4 binding ratios and intensity are altered. Conclusion: At a population level, the linear IgE and IgG4 epitopes of 10 peanut allergens overlap and that increase in IgG4 with OIT results in displacement of IgE binding to both conformational and linear epitopes. Furthermore, it appears as though the increase in IgG4 is more important to achieve desensitization at the 12-month timepoint than the decrease in IgE. This type of knowledge can be useful in the identification of IgE and IgG4-binding allergen and peptide biomarkers that may indicate desensitization or sustained unresponsiveness of allergic individuals to peanut.

Structural and immunologic characterization of Ara h 1, a major peanut allergen.

Allergic reactions to peanuts and tree nuts are major causes of anaphylaxis in the United States. We compare different properties of natural and recombinant versions of Ara h 1, a major peanut allergen, through structural, immunologic, and bioinformatics analyses. Small angle x-ray scattering studies show that natural Ara h 1 forms higher molecular weight aggregates in solution. In contrast, the full-length recombinant protein is partially unfolded and exists as a monomer. The crystal structure of the Ara h 1 core (residues 170-586) shows that the central part of the allergen has a bicupin fold, which is in agreement with our bioinformatics analysis. In its crystalline state, the core region of Ara h 1 forms trimeric assemblies, while in solution the protein exists as higher molecular weight assemblies. This finding reveals that the residues forming the core region of the protein are sufficient for formation of Ara h 1 trimers and higher order oligomers. Natural and recombinant variants of proteins tested in in vitro gastric and duodenal digestion assays show that the natural protein is the most stable form, followed by the recombinant Ara h 1 core fragment and the full-length recombinant protein. Additionally, IgE binding studies reveal that the natural and recombinant allergens have different patterns of interaction with IgE antibodies. The molecular basis of cross-reactivity between vicilin allergens is also elucidated.

Influence of enzymatic hydrolysis on the allergenicity of roasted peanut protein extract.

BACKGROUND: Peanut allergy is recognized as one of the most severe food allergies. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as the soybean, chickpea and lentil. Nevertheless, there are only a few studies carried out with sera from patients with a well-documented allergy. METHODS: Roasted peanut protein extract was hydrolyzed by the sequential and individual action of 2 food-grade enzymes, an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to roasted peanut extract and hydrolyzed samples was evaluated by means of IgE immunoblot, ELISA and 2-dimensional electrophoresis using sera from 5 patients with a clinical allergy to peanuts and anti-Ara h 1, anti-Ara h 2 and anti-Ara h 3 immunoblots. RESULTS: Immunoblot and ELISA assays showed an important decrease of IgE reactivity and Ara h 1, Ara h 2 and Ara h 3 levels in the first 30 min of hydrolyzation with Alcalase. In contrast, individual treatment with Flavourzyme caused an increase in IgE reactivity detected by ELISA at 30 min and led to a 65% inhibition of IgE reactivity at the end of the assay (300 min). Ara h 1 and the basic subunit of Ara h 3 were still recognized after treatment with Flavourzyme for 300 min. CONCLUSION: Hydrolysis with the endoprotease Alcalase decreases IgE reactivity in the soluble protein fraction of roasted peanut better than hydrolysis with the exoprotease Flavourzyme.

Effect of high-moisture extrusion and addition of transglutaminase on major peanut allergens content extracted by three step sequential method.

The effect of high-moisture extrusion (HME) with or without transglutaminase (TGase) on peanut allergen levels and their extractability was studied. A three-stage sequential protein extraction significantly improved the protein recovery in processed samples (extrudate meat analogue); from 5.56 to 18.75 mg/100 mg without TGase, and from 4.59 to 20.82 mg/100 mg with 0.3% TGase. The total major allergen content was reduced by 91% (Ara h 1), 61% (Ara h 2), 60% (Ara h 6), and 55% (Ara h 3). Western-blot analysis of soluble extracts reflected the presence of Ara h 1 and Ara h 2 in significantly lower, indicating a potential reduction in IgE binding. During different processing zones, the most significant reduction in allergenic proteins was in the melting zone. The significant alteration in secondary and tertiary structures as a result of crosslinking shearing and degradation of proteins is likely to lead to allergen reduction.

Structure, Immunogenicity, and IgE Cross-Reactivity among Walnut and Peanut Vicilin-Buried Peptides.

Vicilin-buried peptides (VBPs) from edible plants are derived from the N-terminal leader sequences (LSs) of seed storage proteins. VBPs are defined by a common α-hairpin fold mediated by conserved CxxxCx(10-14)CxxxC motifs. Here, peanut and walnut VBPs were characterized as potential mediators of both peanut/walnut allergenicity and cross-reactivity despite their low (∼17%) sequence identity. The structures of one peanut (AH1.1) and 3 walnut (JR2.1, JR2.2, JR2.3) VBPs were solved using solution NMR, revealing similar α-hairpin structures stabilized by disulfide bonds with high levels of surface similarity. Peptide microarrays identified several peptide sequences primarily on AH1.1 and JR2.1, which were recognized by peanut-, walnut-, and dual-allergic patient IgE, establishing these peanut and walnut VBPs as potential mediators of allergenicity and cross-reactivity. JR2.2 and JR2.3 displayed extreme resilience against endosomal digestion, potentially hindering epitope generation and likely contributing to their reduced allergic potential.

IgE epitopes of Ara h 9, Jug r 3, and Pru p 3 in peanut-allergic individuals from Spain and the US.

Non-specific lipid transfer proteins (LTPs) are well studied allergens that can lead to severe reactions, but often cause oral allergy syndrome in the Mediterranean area and other European countries. However, studies focused on LTP reactivity in allergic individuals from the United States are lacking because they are not considered major allergens. The goal of this study is to determine if differences in immunoglobulin (Ig) E binding patterns to the peanut allergen Ara h 9 and two homologous LTPs (walnut Jug r 3 and peach Pru p 3) between the US and Spain contribute to differences observed in allergic reactivity. Synthetic overlapping 15-amino acid-long peptides offset by five amino acids from Ara h 9, Jug r 3, and Pru p 3 were synthesized, and the intact proteins were attached to microarray slides. Sera from 55 peanut-allergic individuals from the US were tested for IgE binding to the linear peptides and IgE binding to intact proteins using immunofluorescence. For comparison, sera from 17 peanut-allergic individuals from Spain were also tested. Similar IgE binding profiles for Ara h 9, Jug r 3, and Pru p 3 were identified between the US and Spain, with slight differences. Certain regions of the proteins, specifically helices 1 and 2 and the C-terminal coil, were recognized by the majority of the sera more often than other regions of the proteins. While serum IgE from peanut-allergic individuals in the US binds to peptides of Ara h 9 and its homologs, only IgE from the Spanish subjects bound to the intact LTPs. This study identifies Ara h 9, Jug r 3, and Pru p 3 linear epitopes that were previously unidentified using sera from peanut-allergic individuals from the US and Spain. Certain regions of the LTPs are recognized more often in US subjects, indicating that they represent conserved and possible cross-reactive regions. The location of the epitopes in 3D structure models of the LTPs may predict the location of potential conformational epitopes bound by a majority of the Spanish patient sera. These findings are potentially important for development of peptide or protein-targeting diagnostic and therapeutic tools for food allergy.

Manufacturing processes of peanut (Arachis hypogaea) allergen powder-dnfp.

Background: Important components of drug safety, efficacy, and acceptability involve manufacturing and testing of the drug substance and drug product. Peanut flour sourcing/processing and manufacturing processes may affect final drug product allergen potency and contamination level, possibly impacting drug safety, quality, and efficacy. We describe key steps in the manufacturing processes of peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Palforzia®), a drug used in oral immunotherapy (OIT) for the treatment of peanut allergy. Methods: Established criteria for source material must be met for manufacturing PTAH drug product. Degree of roasting was determined with a Hunter colorimeter. Protein/allergen content, identity, potency, safety, and quality of each batch of PTAH drug substance were assessed with a combustion analyzer, allergen-specific Western blot (immunoblotting), ELISA, and HPLC. Contaminants (ie, aflatoxin) were measured by UPLC. Results: Roasting degree beyond "light roast" was associated with variable degrees of protein allergen degradation, or potentially aggregation. Relative potency and amounts of protein allergens showed variability due in part to seasonal/manufacturing variability. Proportion of lots not meeting aflatoxin limits has increased in recent years. Up to 60% of peanut flour source material failed to meet screening selection acceptance criteria for proceeding to drug substance testing, mostly because of failure to meet potency acceptance criteria. Other lots were rejected due to safety (ie, aflatoxin) and quality. Influence of potency variation, within specification parameters, on safety/tolerability observed in trials was considered low, in part due to stringent controls placed at each step of manufacturing. Conclusions: Extensive variability in allergen potency is a critical issue during immunotherapy, particularly during OIT initial dose escalation and up-dosing, as it may result in lack of efficacy or avoidable adverse allergic reactions. Based on EU and US regulatory requirements, the production of PTAH includes manufacturing controls to ensure drug product safety, potency, and quality. For example, although PTAH contains all peanut allergens, each lot has met strict criteria ensuring consistent allergenic potency of Ara h 1, Ara h 2, and Ara h 6. The rigor of PTAH's manufacturing process ensures reliable dose consistency and stability throughout its shelf life.