Recombinant Peanut Allergens Research Articles

Specific features of sensitization to recombinant peanut allergens in children with anaphylaxis

Molecular allergy diagnosis makes it possible not only to identify allergic sensitization to peanut as a whole, but also to establish the specific allergen the specific IgE is synthesized to. The purpose of this study was to establish a relationship between sensitization to certain molecules of peanut and allergy as anaphylaxis after eating the product. An immunofluorescence assay (Immuno CAP 100 Phadia AB, Switzerland) was used to determine specific IgE to whole allergen in the peanut and its components (Ara h1, Ara h2, Ara h3, Ara h8, and h9 Ara). Children diagnosed with peanut-induced anaphylaxis were found to be significantly more sensitized to the recombinant allergens Ara h8 and Ara h9 (p=0.019). The detected combination of sensitization to the recombinant peanut allergen Ara h8 and Ara h9 indicates a high probability that an allergic reaction may manifest as anaphylaxis. Determination of specific IgE against peanut proteins will optimize patient management tactics.

Peanut sensitization pattern in Norwegian children and adults with specific IgE to peanut show age related differences.

BackgroundPeanuts contain potent food allergens and the prevalence of allergy is reported to increase, especially in children. Since peanut sensitization may differ between different geographical regions, we wanted to investigate the sensitization pattern to the individual peanut allergens in a Norwegian population.MethodsCases reported to the Norwegian Food Allergy Register with sera positive to peanut extract were analyzed for specific IgE (sIgE) to the recombinant peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 8 and Ara h 9 and to birch pollen extract. Serum samples negative to the above allergens were analyzed for sIgE to Ara h 6, and sIgE to Pru p 3 in peach were analyzed in sera positive to the cross-reactive allergen Ara h 9.ResultsHighest frequency of sIgE to Ara h 2, often co-sensitized to Ara h 1 and 3, were found in the small children up to 6 years of age. From the age of 6 years, sensitization to Ara h 8 was predominant. The sIgE levels to the storage proteins Ara h 1, 2 and 3 were strongly correlated, as was the sIgE levels to Ara h 8 and birch pollen extract. A low sensitization rate of sIgE to Ara h 9 in young adults was observed, which sIgE levels were very strongly correlated to Pru p 3.ConclusionThe sensitization to peanut allergens in a Norwegian population shows a clear age dependent pattern. The results add to the previously published research on the sensitization patterns of peanut sensitized patients in different geographical areas.

THE CLINICAL ROLE OF COMPONENT DIAGNOSTICS OF PEANUT ALLERGY AMONG CHILDREN WITH ALLERGIC DISEASES

Background. To determine the clinical significance of IgEmediated sensibilization to recombinant peanut allergens. Materials and methods. We examined 30 children with allergic diseases and sensibilization to whole peanut allergen. All children passed through allergological examination, including determination of specific IgE (method of Measurement CAP 100 Phadia, Switzerland) to recombinant peanut allergens: Ara h1, Ara h2, Ara h3, Ara and Ara h8, h9. Results. A wide variation of combinations of sensibilization to various peanut allergens was found. Sensibilization to rAra hi allergen was found in 40% of cases, to rAra h2 - in 36,6%, to rAra h3 - in 33%, to rAra h8 - in 60%, to rAra h9 in 56,6% of cases. Their clinical significance was 36,6% (rAra hi), 26% (rAra h2), 23% (rAra h3), 33% (rAra h8), 36% (rAra h9). Monosensibilization to peanut allergens rAra hi, 8 and 9 was identified in 26,6% of cases. The interrelation of rAra h2 and rAra h8 with atopic dermatitis and rAra h3 with asthma was found. Conclusion. Component diagnostics of peanut allergens has high diagnostic significance in further administration of elimination diets.

Relationship of IgE to basophil phenotypes in peanut-sensitized adults

Relationship of IgE to basophil phenotypes in peanut-sensitized adults

Moving from peanut extract to peanut components: towards validation of component‐resolved IgE tests

Replacement of peanut extracts by recombinant peanut components is an important step in allergy serologic testing. Criteria are needed for the unbiased inclusion of patients into a study to validate such a replacement. Plasma samples from 64 peanut-positive children (42 reactors, 22 nonreactors in a double-blind, placebo-controlled food challenge) were used to compare IgE reactivity to six recombinant peanut allergens with reactivity to natural peanut proteins extracted at neutral or low pH. We tested the hypothesis that poor extractability of Ara h 9 and other basic allergens at neutral pH leads to under-representation of patients with such sensitization. IgE reactivity to the components did not fully explain IgE reactivity to peanut extract in 5 of 32 reactors with IgE to peanut extract ≤100kUA /l. IgE reactivity to components was stronger than to the extract in 11 plasma samples, which was largely due to a low Ara h 8 reactivity of the extract. IgE reactivity to Ara h 9 was much lower than reactivity to other basic proteins, some of which bound IgE well in the RAST, but lost IgE reactivity upon immunoblotting. Conventional peanut extracts are deficient in significant IgE-binding components. The inclusion of patients for a validation study should be based on serology performed with improved peanut reagents to avoid a bias against these under-represented, potentially important allergens. To judge clinical relevance of an allergen, the reagent used for inclusion of patients needs to be efficient in detecting IgE to this component.

Evaluation of IgE Antibodies to Recombinant Peanut Allergens in Patients with Reported Reactions to Peanut

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 kU_A/l, while 100% of the subjects without detectable IgE to these allergens had low peanut-specific IgE levels (<10 kU_A/l). The levels of IgE to rAra h 8, rBet v 1 and birch pollen were highly correlated (r_S = 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.

Peanut allergy: Clinical and immunologic differences among patients from 3 different geographic regions

Peanut allergy affects persons from various geographic regions where populations are exposed to different dietary habits and environmental pollens. We sought to describe the clinical and immunologic characteristics of patients with peanut allergy from 3 countries (Spain, the United States, and Sweden) using a molecular component diagnostic approach. Patients with peanut allergy from Madrid (Spain, n = 50), New York (United States, n = 30), Gothenburg, and Stockholm (both Sweden, n = 35) were enrolled. Clinical data were obtained either from a specific questionnaire or gathered from chart reviews. IgE antibodies to peanut extract and the peanut allergens rAra h 1, 2, 3, 8 and 9, as well as to cross-reactive birch (rBet v 1) and grass (rPhl p 1, 5, 7, and 12) pollen allergens, were analyzed. American patients frequently had IgE antibodies to rAra h 1 to 3 (56.7% to 90.0%) and often presented with severe symptoms. Spanish patients recognized these 3 recombinant peanut allergens less frequently (16.0% to 42.0%), were more often sensitized to the lipid transfer protein rAra h 9 (60.0%), and typically had peanut allergy after becoming allergic to other plant-derived foods. Swedish patients detected rAra h 1 to 3 more frequently than Spanish patients (37.1% to 74.3%) and had the highest sensitization rate to the Bet v 1 homologue rAra h 8 (65.7%), as well as to rBet v 1 (82.9%). Spanish and Swedish patients became allergic to peanut at 2 years or later, whereas the American children became allergic around 1 year of age. Peanut allergy has different clinical and immunologic patterns in different areas of the world. Allergen component diagnostics might help us to better understand this complex entity.

A Novel Immunoassay Using Recombinant Allergens Simplifies Peanut Allergy Diagnosis

Background: Double-blind placebo-controlled food challenge (DBPCFC) is currently considered the gold standard for peanut allergy diagnosis. However, this procedure that requires the hospitalization of patients, mostly children, in specialized centers for oral exposure to allergens may cause severe reactions requiring emergency measures. Thus, a simpler and safer diagnosis procedure is needed. The aim of this study was to evaluate the diagnostic performance of a new set of in vitro blood tests for peanut allergy. Methods: The levels of IgE directed towards peanut extract and recombinant peanut allergens Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 7, and Ara h 8 were measured in 3 groups of patients enrolled at 2 independent centers: patients with proven peanut allergy (n = 166); pollen-sensitized subjects without peanut allergy (n = 61), and control subjects without allergic disease (n = 10). Results: Seventy-nine percent of the pollen-sensitized patients showed IgE binding to peanut, despite their tolerance to peanut. In contrast, combining the results of specific IgE to peanut extract and to recombinant Ara h 2 and Ara h 6 yielded a peanut allergy diagnosis with a 98% sensitivity and an 85% specificity at a positivity threshold of 0.10 kU/l. Use of a threshold of 0.23 kU/l for recombinant Ara h 2 increased specificity (96%) at the cost of sensitivity (93%). Conclusion: A simple blood test can be used to diagnose peanut allergy with a high level of precision. However, DBPCFC will remain useful for the few cases where immunological and clinical observations yield conflicting results.

Immunoglobulin‐E Reactivity to a Glycosylated Food Allergen (Peanuts) Due to Interference With Cross‐Reactive Carbohydrate Determinants in Heavy Drinkers

N-glycans in plant and invertebrate glycoproteins can induce extensive IgE cross-reactivity therefore limiting the specificity of in vitro allergy tests. IgE sensitization to N-glycans (cross-reactive carbohydrate determinants, CCDs) may be increased in heavy drinkers, who therefore show IgE reactivity to aeroallergens, latex, and Hymenoptera venoms. The peanut, a CCD-bearing allergen, is the leading cause of severe food allergic reactions in many populations. To investigate the potential interference of CCDs with determinations of IgE to peanuts in heavy drinkers. We determined IgE to peanuts and IgE to a CCD marker (MUXF(3), the N-glycan from bromelain) in 41 heavy drinkers admitted to the hospital and 54 healthy controls. None of the participants reported symptoms of peanut allergy. In cases with positive (>or=0.35 kU/l) IgE to peanuts, we performed inhibition assays with a neoglycoprotein consisting of MUXF(3) molecules coupled to bovine serum albumin (MUXF(3)-BSA) and a similar neoglycoprotein lacking xylose and fucose (MM-BSA). In the same cases, we screened for IgE to a panel of recombinant nonglycosylated peanut allergens. SDS-PAGE immunoblotting and inhibition assays were performed in selected cases. The prevalence of positive IgE to peanuts was 22 and 3.7% in heavy drinkers and healthy controls, respectively (p < 0.001). Peanut-IgE positivity was closely related to the presence of IgE to CCDs. In most (8/9) heavy drinkers with positive IgE to peanuts, reactivity was inhibited by preincubation with MUXF(3)-BSA, but not with MM-BSA. IgE binding to multiple bands on immunoblotting studies was also inhibited by MUXF(3)-BSA preincubation. IgE to nonglycosylated recombinant peanut allergens was uniformly negative. Heavy drinking is associated with clinically asymptomatic IgE reactivity to peanuts, a relevant food allergen, in relation to CCD interference.

Predictive value of skin prick tests using recombinant allergens for diagnosis of peanut allergy

Current diagnosis of peanut allergy relies on natural extracts that lack standardization. Recombinant DNA technology allows production of pure biochemically characterized proteins. Their usefulness for peanut allergy diagnosis is not established. This study aimed to evaluate the diagnostic value of the 3 major recombinant peanut allergens. Recombinant (r) Ara h 1, rAra h 2, and rAra h 3 were produced according to the recommendations of good manufacturing practice for recombinant allergens. Skin prick tests (SPTs) and IgE ELISA assays were performed in 30 patients with peanut allergy and 30 control subjects without food allergy: 15 nonatopic and 15 sensitized to birch pollen. Disease severity was graded by clinical scoring. All patients with peanut allergy showed positive SPT results to rAra h 2; 40% reacted with rAra h 1 and 27% with rAra h 3. No control subjects reacted with any of the recombinant allergens. Monosensitization to rAra h 2 was observed in 53% of patients. Neither SPT size nor levels of specific IgE were correlated with the disease severity. However, patients with monosensitization to rAra h 2 had a significantly lower severity score than polysensitized subjects and a lower level of specific IgE against peanut extract and rAra h 2. Skin prick tests to individual recombinant peanut allergens appear to be a safe and effective diagnostic tool. Cosensitization to rAra h 2 and rArah 1 and/or rAra h 3 is predictive of more severe reactions. Recombinant peanut allergens can be used by SPTs for diagnosis and evaluation of allergy severity.

Purification and immunoglobulin E‐binding properties of peanut allergen Ara h 6: evidence for cross‐reactivity with Ara h 2

IgE-binding peanut proteins smaller than 15 kDa were previously identified as potential allergens in the majority of our peanut allergic population. To characterize the novel allergen in order to determine whether it was similar to one of the thus far identified recombinant peanut allergens (Ara h 1-7). An IgE-binding protein of <15 kDa was purified and identified via N-terminal sequencing. Its IgE-binding properties were investigated using immunoblotting, basophil degranulation, and skin prick testing. Possible cross-reacting epitopes with other peanut allergens were studied using IgE-immunoblotting inhibition. The purified protein is a monomeric protein with a molecular weight of 14,981 Da as determined using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. The amino acid sequence of the first 39 N-terminal residues is identical to that of Ara h 6, indicating that the allergen is Ara h 6. It is recognized by 20 out of 29 peanut-allergic patients on IgE-immunoblot, and its potent biological functionality is demonstrated by the degranulation of basophils, even at concentrations below 10 pg/mL, and by positive skin prick reactions. Ara h 6 has homology to Ara h 2, especially in the middle part and at the C-terminal part of the protein. Almost complete inhibition of IgE-Ara h 6 interaction with Ara h 2 demonstrates that at least part of the epitopes of Ara h 6 are cross-reactive with epitopes on Ara h 2. Peanut-derived Ara h 6 is a biologically active allergen recognized by the majority of our peanut-allergic patient population and can be considered a clinically relevant peanut allergen.

Purification and IgE-binding properties of peanut allergen Ara h 6: Evidence for cross-reactivity with Ara h2

Rationale IgE-binding peanut proteins smaller than 15 kDa were previously identified as potential allergens in the majority of a peanut allergic population. We characterized the novel allergen and determined whether it was similar to one of the thus far identified recombinant peanut allergens (Ara h1 -7). Methods The IgE-binding protein of <15 kDa was purified and identified via N-terminal sequencing. Its IgE-binding properties were investigated using ELISA, immunoblotting and basophil histamine release. Possible cross-reacting epitopes with other peanut allergens were studied using IgE-immunoblotting inhibition. Results The purified protein is a monomeric protein with a molecular weight of 14,981 Da as determined with MALDI-TOF mass-spectroscopy. The amino acid sequence of the first 39 N-terminal residues is identical to that of Ara h6, indicating that the allergen is Ara h6. It is recognized by 20 out of 30 peanut-allergic patients on IgE-immunoblot, and its potent biological functionality is demonstrated by the release of histamine from basophils, even at concentrationsbelow 10 pg/ml. Ara h6 has homology with Ara h2, especially in the mid part and the C-terminal part of the protein. Almost complete inhibition of IgE-Ara h6 interaction with Ara h2 demonstrates that at least part of the epitopes of Ara h6 is cross-reactive with epitopes on Ara h2. The partial inhibition of Ara h2-IgE interaction with Ara h6 indicates that next to shared epitopes, unique IgE-binding epitopes on Ara h2 exists. Conclusions Peanut-derived Ara h 6 is a biologically active allergen recognized by the majority of our patient population.

Use of Modified BL21(DE3) Escherichia coli Cells for High-Level Expression of Recombinant Peanut Allergens Affected by Poor Codon Usage

We previously cloned a panel of peanut allergens by phage display technology. Examination of the codons used in these sequences indicated that most of the cDNAs contain an excess of the least used codons in Escherichia coli, namely AGG/AGA, that correspond to a minor tRNA, the product of the dnaY gene. To achieve high-level expression of the peanut allergens, the cDNAs were subcloned into an expression vector of the pET series (Novagen) in order to produce (His)(10)-tagged fusion proteins in conventional E. coli BL21(DE3) cells. The peanut allergens Ara h 1, Ara h 2, and Ara h 6 with an AGG/AGA codon content of 8-10% were only marginally expressed, whereas the peanut profilin Ara h 5, with an AGG/AGA codon content of only 0.8%, was efficiently expressed in these cells. Hence, by using modified BL21(DE3) E. coli cells, namely BL21-CodonPlus(DE3)-RIL cells (Stratagene) with extra copies of E. coli argU, ileY, and leuW tRNA genes, it was possible to attain high-level expression of the proteins affected by rare codon usage. IPTG-induced expression of several recombinant peanut allergens, such as Ara h 1, Ara h 2, and Ara h 6, was greatly increased in these special cells compared to the expression yield achieved by conventional E. coli hosts. The purification of the soluble and the insoluble fraction of Ara h 2 was performed by metal-affinity chromatography and yielded a total of about 30 mg (His)(10)-tagged recombinant protein per liter of culture of transformed BL21(DE3)CodonPlus-RIL cells. This is over 100 times more than achieved by production of Ara h 2 in conventional BL21(DE3) cells.