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.

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.

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.

Epitopes with similar physicochemical properties contribute to cross reactivity between peanut and tree nuts.

Many individuals with peanut (PN) allergy have severe reactions to tree nuts (TN) such as walnuts or cashews. Although allergenic proteins in TN and PN have overall low identity, they share discrete sequences similar in physicochemical properties (PCP) to known IgE epitopes. Here, PCP-consensus peptides (cp, 13 aa and 31 aa) were identified from an alignment of epitope rich regions of walnut vicilin, Jug r 2, leader sequence (J2LS) and cross-reactive epitopes in the 2S albumins of peanut and synthesized. A peptide similarity search in the Structural Database of Allergenic Proteins (SDAP) revealed a network of peptides similar (low property distance, PD) to the 13 aa cp (13cp) in many different plant allergens. Peptides similar to the 13cp in PN and TN allergens bound IgE from sera of patients allergic to PN and TN in peptide microarray analysis. The 13cp was used to produce a rabbit consensus peptide antibody (cpAB) that detected proteins containing repeats similar to the 13cp in western blots of various nut extracts, in which reactive proteins were identified by mass spectrometry. The cpAB bound more specifically to allergens and nut extracts containing multiple repeats similar to the 13 cp, such as almond (Pru du 6), peanut (Ara h 2) and walnut (Jug r 2). IgE binding to various nut extracts is inhibited by recombinant J2LS sequence and synthetic 31cp. Thus, several repeated sequences similar to the 13cp are bound by IgE. Multiple similar repeats in several allergens could account for reaction severity and clinically relevant cross-reactivity to PN and TN. These findings may help improve detection, diagnostic, and therapeutic tools.

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.

Purification and Characterization of Pathogenesis Related Class 10 Panallergens.

Oral allergy syndrome (OAS) describes an allergic reaction where an individual sensitized by pollen allergens develops symptoms after eating certain foods. OAS is caused by cross-reactivity among a class of proteins ubiquitous in plants called pathogenesis related class 10 (PR-10) proteins. The best characterized PR-10 protein is Bet v 1 from birch pollen and its putative function is binding hydrophobic ligands. We cloned a subset of seven recombinant PR-10 proteins from pollens, peanuts, and hazelnuts and developed a standard purification method for them. Immunoglobulin E (IgE) binding of purified PR-10 proteins was analyzed by ImmunoCAP ISAC microarray and enzyme-linked immunosorbent assays (ELISAs) with sera from allergic patients. We investigated the binding activities of PR10s by testing 16 different ligands with each protein and compared their secondary structures using circular dichroism (CD). The PR-10s in this study had very similar CD spectra, but bound IgE with very different affinities. All seven proteins showed a similar pattern of binding to the polyphenol ligands (resveratrol, flavonoids, and isoflavones) and variable binding to other potential ligands (fatty acids, sterols, and plant hormones). We suggest our protocol has the potential to be a near-universal method for PR-10 purification that will facilitate further research into this important class of panallergens.

Contribution of Chemical Modifications and Conformational Epitopes to IgE Binding by Ara h 3.

Roasting is known to change the allergenic properties of peanuts. To study these observations at a molecular level, the relationship of IgE binding to the structure of Ara h 3 from raw and roasted peanuts was assessed. Ara h 3 (A3) was purified from raw (R), light roast (LR) and dark roast (DR) peanuts, the purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the secondary structures were compared with circular dichroism (CD) spectroscopy. In order to understand the contribution of structure to IgE binding, the R A3 was partially denatured (PD) by heat treatment (65 °C for 2 h), subjected to CD spectroscopy and IgE spot blot analysis with sera from peanut- allergic individuals. While we observed that the secondary structure of purified A3 from R and LR peanut in solution was affected by the reduction of disulfide bonds and heat treatment when purified from the peanut following the roasting process, only small alterations were seen in the secondary structure. The purified LR A3 bound higher levels of IgE than the RA3. CD spectroscopy of PD A3 revealed a reduction in the percentage of alpha helices, and serum IgE binding. Therefore, while A3 purified from roasted peanuts did not show significant changes in secondary structure, it showed higher IgE binding than R A3. Therefore, the higher IgE binding to LR A3 was more likely to be due to chemical modifications than structural changes. However, a decrease in the IgE binding was seen if R A3 was deliberately unfolded, indicating that the structure played an important role in IgE binding to A3.

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.

Influence of enzymatic hydrolysis on the allergenic reactivity of processed cashew and pistachio.

Cashew and pistachio allergies are considered a serious health problem. Previous studies have shown that thermal processing, pressurization and enzymatic hydrolysis may reduce the allergenic properties of food by changing the protein structure. This study assesses the allergenic properties of cashew and pistachio after thermal treatment (boiling and autoclaving), with or without pressure (autoclaving), and multiple enzymatic treatments under sonication, by SDS-PAGE, western blot and ELISA, with serum IgE of allergic individuals, and mass spectroscopy. Autoclaving and enzymatic hydrolysis under sonication separately induced a measurable reduction in the IgE binding properties of pastes made from treated cashew and pistachio nuts. These treatments were more effective with pistachio allergens. However, heat combined with enzymatic digestion was necessary to markedly lower IgE binding to cashew allergens. The findings identify highly effective simultaneous processing conditions to reduce or even abolish the allergenic potency of cashew and pistachio.

Allergenicity attributes of different peanut market types.

Four different market classes of peanut (Runner, Virginia Spanish, and Valencia) are commonly consumed in Western countries, but for some consumers peanuts are a main cause of food-induced anaphylaxis. Limited information is available on the comparative allergenicity of these distinct market classes. The aim of this study was to compare allergenicity attributes of different peanut cultivars. The protein content and protein profiles were highly comparable for all tested cultivars. All cultivar samples contained the major allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as assessed by SDS-PAGE and RP-HPLC, although some minor differences in major allergen content were found between samples. All samples were reactive in commercial ELISAs for detection and quantification of peanut protein. IgE-binding potency differed between samples with a maximum factor of 2, indicating a highly comparable allergenicity. Based on our observations, we conclude that peanuts from the main market types consumed in Western countries are highly comparable in their allergenicity attributes, indicating that safety considerations with regard to peanut allergy are not dependent on the peanut cultivar in question.

Allergenic properties and differential response of walnut subjected to processing treatments.

The aim of this study was to investigate changes in walnut allergenicity after processing treatments by in vitro techniques and physiologically relevant assays. The allergenicity of walnuts subjected to high hydrostatic pressure and thermal/pressure treatments was evaluated by IgE-immunoblot and antibodies against walnut major allergen Jug r 4. The ability of processed walnut to cross-link IgE on effector cells was evaluated using a rat basophil leukaemia cell line and by skin prick testing. Susceptibility to gastric and duodenal digestion was also evaluated. The results showed that walnuts subjected to pressure treatment at 256 kPa, 138 °C, were able to diminish the IgE cross-linking capacity on effector cells more efficiently than high pressure treated walnuts. IgE immunoblot confirmed these results. Moreover, higher susceptibility to digestion of pressure treated walnut proteins was observed. The use of processed walnuts with decreased IgE binding capacity could be a potential strategy for walnut tolerance induction.

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.

Pine nut allergy: clinical features and major allergens characterization.

SCOPE: The aims of this study were to evaluate IgE-mediated hypersensitivity to pine nut with details of clinical reactions and to characterize major pine nut allergens. METHODS AND RESULTS: The study included ten consecutive teenagers and adults diagnosed with IgE-mediated clinical allergy to pine nut. Two major pine nut allergens were purified and identified and the secondary structures and susceptibility to digestion were characterized. Severe reactions represent 80% of allergic reactions to pine nut in this study. Moreover, 70% of the patients were monosensitized to this nut. Two major allergens with molecular weights of 6 and 50 kDa were purified and identified as albumin and vicilin, respectively. The 6 kDa protein (albumin), rich in α-helix content, was far more stable to peptic and tryptic digestion as compared with 50 kDa protein (vicilin), which was quickly broken down. The secondary structure of the purified 50 kDa protein showed 41% ß-sheet, 5% α-helix, and 54% random coil and/or loops. CONCLUSION: Eighty percent of allergic reactions to pine nut in the ten patients included in this study were severe. Most patients (70%) were monosensitized to this nut. Two major allergens with molecular weights of 6 and 50 kDa were purified and identified as albumin and vicilin, respectively.

Ara h 1 structure is retained after roasting and is important for enhanced binding to IgE.

SCOPE: Ara h 1 from roasted peanut binds higher levels of serum immunoglobulin E than raw peanuts and this is likely due to the Maillard reaction. While Ara h 1 linear IgE epitopes have been mapped, the presence and importance of structural epitopes is not clear. METHODS AND RESULTS: Mass spectrometry, immunoblot, ELISA, circular dichroism (CD), and structural analysis were used to compare structural and subsequent IgE-binding differences in Ara h 1 purified from raw (N) and roasted peanuts (R) and denatured Ara h 1 (D). Although N and R had similar CD spectra, the latter bound significantly more IgE. Decreased IgE binding was seen with the loss of secondary structure. This same IgE-binding pattern [R > N > D] was seen for the sera of ten peanut allergic patients. While the majority of linear epitopes are located on surface and structured regions of Ara h 1, our study shows that conformational epitopes of Ara h 1 bind better to IgE than linear epitopes. CONCLUSION: Enhanced IgE binding to roasted Ara h 1 could be due to alterations such as chemical modifications to individual amino acids or increased epitope exposure. IgE binding is significantly reduced with loss of structure.

Enzymatic treatment of peanut kernels to reduce allergen levels.

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.

Differences among heat-treated, raw, and commercial peanut extracts by skin testing and immunoblotting.

BACKGROUND: Peanut allergenicity has been reported to be influenced by heat treatment, yet the commonly available extracts for skin prick testing (SPT) are derived from raw extracts. OBJECTIVE: To assess the effect of heat treatment on the SPT reactivity and specific IgE binding to peanut. METHODS: Three commercial extracts and 3 laboratory-prepared extracts, including raw, roasted, and boiled, were used for SPT in 19 patients with suspected peanut allergy and in 4 individuals who eat peanut without any symptoms. Serum samples were obtained to measure total IgE in addition to specific IgE binding to the study extracts by immunoblotting. Peanut allergy was confirmed with challenge test unless the individual had a convincing history of a severe reaction. RESULTS: Eleven study participants were considered peanut allergic based on a strong history or positive challenge test result. SPT with the prepared and commercial reagents showed that the boiled extract had the highest specificity (67% vs 42%-63% for the other extracts). The prepared extracts showed similar SPT sensitivity (81%). Three patients with a history of severe reaction and elevated specific IgE levels to peanut to the 3 study extracts had variable SPT reactivity to 1 or more of the commercial extracts. IgE binding to Ara h 2 was found in nearly all patients, regardless of their clinical reactivity. CONCLUSIONS: None of the extracts tested showed optimal diagnostic reliability regarding both sensitivity and specificity. Perhaps testing should be performed with multiple individual extracts prepared by different methods.

Effects of boiling on the IgE-binding properties of tropomyosin of shrimp (Litopenaeus vannamei).

The thermal stability and IgE binding of raw and boiled shrimp extracts and the tropomyosins (TM) have not been reported. In this study, we compare the stability of raw and boiled shrimp TM of Litopenaeus vannamei and evaluate how boiling may alter the allergenicity of L. vannamei. Extracts were prepared from raw and boiled shrimp and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis. The IgE-binding of the extracts was determined by western-blot and competitive inhibition enzyme-linked immunosorbent assay (iELISA). The TM was then purified from raw and boiled shrimp, the secondary structures analyzed by circular dichroism (CD) spectroscopy, and the IgE binding compared by slot blot analysis. The soluble protein content decreased and the higher molecular weight proteins increased in the extracts from boiled versus raw shrimp. Similar IgE binding characteristics were seen by extracts when using western blot analysis. Although iELISA results showed that extracts from raw shrimp bound higher IgE than extracts from boiled shrimp, dot-blot assay demonstrates higher IgE binding to purified TM from boiled shrimp than raw shrimp. The purified TM had a typical alpha-helical secondary structure and the stability of boiled TM was lower than that of raw TM. Extracts from boiled shrimp produce lower IgE binding than extracts from raw shrimp, which suggest that boiling can be used as a tool in attempting to reduce shrimp allergenicity. However, the purified TM from boiled shrimp, which shows enhanced IgE binding over that of raw shrimp, may be a more effective antigen in diagnosing shrimp allergy through immunoassay.

Processing can alter the properties of peanut extract preparations.

As peanut allergy is an increasing public health risk, affecting over 1% of the United States and United Kingdom school children, it is important that methods and reagents for accurate diagnosis of food allergy and detection of allergenic foods are reliable and consistent. Given that most current experimental, diagnostic, and detection tests rely on the presence of soluble allergens in food extracts, we investigated the effects of thermal processing on the solubility and IgE binding of the major peanut allergens, Ara h 1 and Ara h 2. The soluble and insoluble fractions of peanuts that were boiled, fried, and roasted were subjected to electrophoresis and Western blot analysis using anti-Ara h 1 and anti-Ara h 2 antibodies and serum IgE from peanut allergic individuals. Overall protein solubility is reduced with processing and IgE binding increases in the insoluble fractions, due mostly to the increase in the amount of insoluble proteins, with increased time of heating in all processes tested. Therefore, it can be concluded that thermal processing of peanuts alters solubility, and the differences in protein solubility within various extract preparations may contribute to inconsistent skin prick test and immunoassay results, particularly when nonstandardized reagents are used.

Reduction of IgE binding and nonpromotion of Aspergillus flavus fungal growth by simultaneously silencing Ara h 2 and Ara h 6 in peanut.

The most potent peanut allergens, Ara h 2 and Ara h 6, were silenced in transgenic plants by RNA interference. Three independent transgenic lines were recovered after microprojectile bombardment, of which two contained single, integrated copies of the transgene. The third line contained multiple copies of the transgene. Ara h 2 expression was significantly suppressed in all three lines, whereas Ara h 6 was reduced in two lines. Expression of peanut allergens Ara h 1 and Ara h 3 was not noticeably affected. Significant reduction of human IgE binding to Ara h 2 and Ara h 6 also was observed. Seed weight and germination data from transgenic and nontransgenic segregants showed no significant differences. Data collected from in vitro Aspergillus flavus infection indicate no significant difference in fungal growth between the transgenic lines and the nontransgenic controls. These data suggest that silencing Ara h 2 and Ara h 6 is a feasible approach to produce hypoallergenic peanut.