Patients with anaphylaxis to pea can have peanut allergy caused by crossreactive IgE to vicilin (Ara h 1)

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1 Patients with to pea can have peanut allergy caused by crossreactive IgE to vicilin (Ara h 1) Marjolein Wensing, MD, a André C. Knulst, MD, a Sander Piersma, PhD, b,c Francesca O Kane, MSc, b,c Edward F. Knol, PhD, a and Stef J. Koppelman,PhD a,b,c Utrecht, Zeist, and Wageningen, The Netherlands Background: Serologic cross-reactivity among legumes has been described; however, it is rarely clinically significant. In this study 3 patients with a history of to pea are described who subsequently had symptoms after ingestion of peanut. Objective: We investigated whether the peanut-related symptoms were due to cross-reactivity between pea and peanut proteins. Methods: Peanut-related symptoms were documented according to case history or double-blind, placebo-controlled food challenge results. Skin prick tests were performed, and specific IgE levels were determined for pea and peanut with the CAP system FEIA. IgE-binding proteins in pea and peanut were identified by using immunoblot analysis. Cross-reactivity was studied by means of immunoblot and ELISA inhibition studies with whole extracts and purified allergens. Results: Peanut-related symptoms consisted of oral symptoms in all patients, with additional urticaria and dyspnea or angioedema in 2 patients. All patients had a positive skin prick test response and an increased IgE level to pea and peanut. Immunoblotting revealed strong IgE binding to mainly vicilin in pea extract and exclusively to Ara h 1 in crude peanut extract. Immunoblot and ELISA inhibition studies with crude extracts, as well as purified proteins, showed that IgE binding to peanut could be inhibited by pea but not or only partially the other way around. Conclusion: Clinically relevant cross-reactivity between pea and peanut does occur. Vicilin homologues in pea and peanut (Ara h 1) are the molecular basis for this cross-reactivity. (J Allergy Clin Immunol 2003;111:420-4.) Key words: Pea, peanut, vicilin, Ara h 1, cross-reactivity The prevalence of food allergy is estimated to be approximately 2% in adults and 8% in children. 1,2 The most common offending foods are milk, egg, peanut, soybean, tree nuts, crustaceans, and fish. 2 Soybean and From a the Department of Dermatology/Allergology, University Medical Centre Utrecht, Utrecht; b the Protein Technology Department, TNO Nutrition and Food Research Institute, Zeist; and c the Centre for Protein Technology, TNO-WU, Wageningen. Supported by TNO Nutrition and Food Research. Received for publication June 7, 2002; revised August 24, 2002, and October 11, 2002; accepted for publication October 16, Reprint requests: Marjolein Wensing, MD, Department of Dermatology/Allergology G02.124, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands Mosby, Inc. All rights reserved /2003 $ doi: /mai Abbreviation used DBPCFC: Double-blind, placebo-controlled food challenge peanut are members of the legume family and are often recognized as allergens. 3-6 Adverse reactions to other legumes, however, hardly have been described. There are a few studies reporting allergic reactions to chickpea, lentil, and lupine As observed in earlier studies, patients with peanut allergy have extensive serologic cross-reactivity among members of the legume family. 11,12 However, clinically significant cross-reactivity among legumes appeared to be rare Some reports do mention adverse reactions to soybean, lupine, and pea in patients with peanut allergy. 7,8,13,16 In Mediterranean areas lentils are most commonly implicated in food allergy frequently associated with symptoms after ingestion of chickpeas. 9 However, only one study, by Moneret- Vautrin et al, 8 actually confirmed lupine-related symptoms caused by cross-reactivity with peanut. Pea allergy is rare and therefore not extensively investigated, resulting in limited information on pea allergens. Malley et al 17,18 demonstrated that only the albumin fraction produced positive skin test results in 10 pea-sensitive subjects, and they later purified a green pea allergen of 1.8 kd. Sequence identity among legume proteins has been described previously. Burks et al 19 described a homology of 60% to 65% in amino acid sequences among vicilin-like proteins (seed storage proteins) in pea and peanut (Ara h 1), and recently, sequence similarity between glycinins in peanut (Ara h 3), soybean, and pea of 62% to 72% was described. 20,21 This report describes 3 patients with severe pea-related symptoms who also had peanut-related symptoms. The aim of this study was to clarify potential cross-reactivity between these 2 legumes and to identify the allergens involved. METHODS Patients Three patients with a history of severe allergic reactions after ingestion of pea who had peanut-related symptoms were investigated in this study. Specific IgE levels to pea and peanut and total IgE

2 J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2 Wensing et al 421 FIG 1. SDS-PAGE and CBB stain: immunoblotting and blot inhibition with serum of patient 3. Lanes 1 to 4, CBB stain of crude pea and peanut extract and vicilin and Ara h 1; lanes 5 to 8, immunoblotting of crude extracts and the purified allergens; lanes 9 to 12, IgE binding to vicilin and Ara h 1 inhibited by using 2 concentrations of vicilin; lanes 13 to 16, IgE binding to vicilin and Ara h 1 inhibited by using 2 concentrations of Ara h 1. levels were determined by using the CAP system FEIA (Pharmacia & Upjohn, Uppsala, Sweden). Skin prick tests with commercial pea and peanut extracts (ALK-Abelló, Nieuwegein, The Netherlands) were performed and recorded as described by Dreborg and Frew. 22 Detailed histories of pea- and peanut-related symptoms were obtained. For one patient, peanut-related symptoms were less convincing, which made a double-blind, placebo-controlled food challenge (DBPCFC) necessary. In short, increasing doses of peanut meal (30 µg-1 g of peanut protein) hidden in mashed potatoes or oatmeal porridge randomly interspersed with an equal amount of placebo doses were administered to the patient. An interval of 30 minutes was allowed when no reactions occurred. A challenge result was regarded as positive when objective reactions occurred or when subjective reactions occurred repeatedly (3 times or more) after active doses. Crude protein extracts Crude protein extracts from pea and peanut were prepared by mixing 1 g of raw ground pea or peanut with 20 ml of 50 mmol/l Tris, ph 8.2, for 2 hours. Mixtures were centrifuged (3000g for 10 minutes) to remove insoluble parts. Protein content was measured by using Bradford analysis, with BSA as a standard. 23 Purified proteins Ara h 1 was purified generally as described previously 24,25 with minor modifications and was essentially pure (>95%), as judged by using a densitometer scan of an SDS-PAGE gel stained with Coomassie Brilliant Blue (Fig 1, lane 4). The N-terminal sequence was determined and appeared to be identical to the earlier published sequence of Ara h Purified Ara h 1 was stored at 80 C until use. Pea vicilin was purified from raw peas of the Solara variety (Pisum sativum) by using a nondenaturing fractionation procedure according to the method of Bora et al. 26 Purified vicilin was analyzed on SDS-PAGE and showed main bands at 50, 33, 30, and 20 kd (Fig 1, lane 3), which was similar to results with earlier published band patterns. 27,28 Purified vicilin was freeze-dried and stored at 20 C until use. SDS-PAGE, immunoblotting, and immunoblot inhibition SDS-PAGE was performed by using standard equipment (Bio- Rad, Hercules, Calif) with 15% acrylamide gels (15 10 cm). Per lane, 7 µg of crude protein extracts and 1.6 µg of the purified proteins was used. Gels were stained with Coomassie Brilliant Blue R- 250 dissolved in destaining solution (10% HAc [vol/vol] and 5% methanol [vol/vol] in water). After destaining, gels were scanned with an ImageMaster DTS (Pharmacia & Upjohn). For Western blotting, SDS-PAGE gels were prepared as described above, and the separated proteins were transferred to polyvinyldifluoride sheets (Immobilon-P; Millipore Corp, Bedford, Mass) by using standard techniques. Membranes were blocked with 3% BSA in wash buffer (50 mmol/l Tris, ph 7.5, containing 0.1% BSA and 0.1% Tween-20) for 1 hour at room temperature. Patients sera were diluted 1:120 in wash buffer and applied to the membrane (overnight at room temperature). In case of inhibition experiments, increasing concentrations of Ara h 1 or vicilin ( and 2-10 µg/ml, respectively) were added to the patients serum 1 hour before application to the blot membrane. Bound IgE was detected by using a commercial antihuman IgE conjugated to peroxidase (Diagnostic Product Company, Los Angeles, Calif) and a subsequent staining reaction for peroxidase activity by using the ECL technique. Between each step, blot membranes were washed thoroughly 5 times with wash buffer. Nonspecific binding of the anti-ige antibody conjugate was measured in a similar blotting procedure, omitting the incubation step with patient sera, and appeared to be negligible.

3 422 Wensing et al J ALLERGY CLIN IMMUNOL FEBRUARY 2003 TABLE I. Patient characteristics CAP Age of Peanut- CAP Age of Total Other legumes Patient Pea-related SPT pea onset related SPT peanut onset IgE that caused no. Sex Age (y) symptoms pea (ku/l) (y) symptoms peanut (ku/l) (y) (ku/l) symptoms 1 F 40 Os, urt, dys, ND >100 3 Os, urt, dys Mp, kb, hb gi, rc, shock 2 F 32 Os, dys, rc Os (confirmed Le, fb, gb, bs, by DBPCFC) bb, mp 3 M 28 Os, urt, dys, ND 70 3 Os, ae ND shock Os, Oral symptoms; urt, urticaria; dys, dyspnea; gi, gastrointestinal symptoms; rc, rhinoconjunctivitis; ND, not done; mp, marrow pea; kb, kidney bean; hb, haricot bean; le, lentils; fb, French beans; gb, green beans; bs, bean sprouts; bb, broad beans; ae, angioedema. ELISA techniques ELISA techniques were based on an earlier report about the Ara h 1 IgE interaction. 29 Plates (96-well) were coated with 10 µg/ml Ara h 1 or pea vicilin by using PBS as a coating buffer. Subsequently, wells were blocked with BSA (1% in PBS containing 0.1% Tween-20, block buffer) to diminish the nonspecific binding. Patients sera were diluted in block buffer, and serum dilutions were incubated on the allergen-coated ELISA plates for 2 hours at 37 C. In inhibition experiments patient serum was preincubated with inhibitor in increasing concentrations (0-100 µg/ml) for 1 hour at room temperature. These mixtures were transferred to the allergencoated ELISA plates and incubated for 2 hours at 37 C. Bound IgE was detected by using an anti-human IgE antibody conjugated to horseradish peroxidase (Diagnostic Product Company) and is expressed as A 490. Between each step, plates were washed 5 times with PBS containing 0.1% Tween-20. Nonspecific reactivity was studied by using serum from a nonallergic individual and appeared to be negligible. Uncoated plates incubated with patient serum did not bind IgE either, indicating that nonspecific binding of IgE to plastic was negligible too. RESULTS Patients Patient characteristics are summarized in Table I. Pearelated symptoms consisted of symptoms of itching and tingling of the oral cavity or lips and dyspnea in all patients. Two patients (nos. 1 and 3) experienced additional symptoms, such as generalized urticaria and cardiovascular symptoms, resulting in faintness. This required attendance at an emergency department more than once. Ingestion of peanut also induced oral symptoms in all patients: in patient 1 this was accompanied by generalized urticaria and dyspnea and in patient 3 by severe angioedema, both requiring medication, such as β-agonists or antihistamines. Patient 2 had oral symptoms exclusively that were confirmed with a DBPCFC. She reported itching and tingling of the oral cavity from a dose of 100 mg of peanut protein (about two thirds of a peanut). This also occurred after the 2 following doses of 300 and 1000 mg but not after placebo doses. All patients had severe allergic reactions to pea before the development of peanut-induced reactions. Two patients reported additional symptoms to other members of the legume family. All patients frequently had allergic reactions caused by accidental ingestion of legumes hidden in prepackaged foods, such as bread and snacks. Identification of allergens Immunoblotting was performed with crude protein extracts from pea and peanut to investigate the pea and peanut allergens recognized by IgE of these patients. Fig 1 (lane 5) shows IgE binding with serum of patient 3 to crude pea extract. IgE-binding protein bands appeared to have molecular weights of 50 kd, 33 kd, 30 kd, between 20 and 30 kd, and 20 kd or less. Such a pattern is indicative for vicilin. 27,28 Indeed, immunoblotting with purified vicilin, as shown in Fig 1, lane 7, demonstrated similar IgE-binding bands. Yet 2 unidentified IgE-binding bands (35 and <14 kd) in crude pea extract could not be explained by IgE to vicilin. Fig 1 also shows IgE binding to crude peanut extract. Interestingly, only one band stained clearly for IgE. The molecular weight of this band is approximately 65 kd, which is indicative of Ara h 1. 19,24,25 This was further confirmed by immunoblotting with purified Ara h 1 (Fig 1, lane 8). Weak IgE binding was also detected to a protein band of approximately 50 kd. Because this band is also present in purified Ara h 1, this binding might be due to a proteolytic fragment of Ara h 1. Taken together, IgE binding to vicilins in both pea and peanut (Ara h 1) could be detected in this patient. Similar recognition profiles were demonstrated by using sera of patients 1 and 2 (not shown), although the Ara h 1 band for patient 2 was less intense, possibly because of a low peanut-specific IgE titer (Table I). Assessment of cross-reactivity of individual pea and peanut allergens Cross-reactivity between the homologous vicilin storage proteins of the legume family was studied by using IgE-binding inhibition studies with purified pea vicilin and Ara h 1. Fig 1 shows the IgE reactivity for patient 3 under different conditions. Preincubations with increasing concentrations of vicilin showed complete inhibition of IgE binding to both vicilin and Ara h 1. Furthermore, the serum was preincubated with 150 or 500 µg/ml Ara h 1. IgE binding to vicilin was still detectable with approximately the same intensity as in the control situation, even with the highest concentration of Ara h 1. The binding to Ara h 1 itself was only partially inhibited by 150 µg/ml Ara h 1 but was inhibited in a more pronounced manner by 500 µg/ml Ara h 1.

4 J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2 Wensing et al 423 Inhibition of IgE binding was studied in an inhibition ELISA to further substantiate this cross-reactivity and to obtain more quantitative results. Sera were preincubated with various concentrations of purified pea vicilin or Ara h 1. Fig 2 demonstrates data of patient 3. Fig 2, A, shows that IgE binding to vicilin can be inhibited by vicilin itself, whereas the vicilin-ige interaction could not be inhibited by Ara h 1. Fig 2, B, shows that IgE binding to Ara h 1 could be inhibited by high concentrations of Ara h 1 itself, but the inhibition with vicilin appeared to be far more potent. Table II summarizes inhibitory concentration of 50% values for IgE inhibition with pea vicilin and Ara h 1 in all 3 patients. In all patients IgE binding to vicilin could be inhibited by vicilin itself but not by Ara h 1, and the IgE binding to Ara h 1 could be inhibited by Ara h 1 itself but far more potently by vicilin. A B DISCUSSION Three patients with histories of severe after ingestion of pea were investigated regarding their peanutrelated symptoms. In one patient peanut-related symptoms were confirmed by means of DBPCFC. The other 2 patients were not challenged with peanut because they had convincing histories of peanut (Table I). Looking at the specific IgE levels, skin prick test results, and the course of development of food-related symptoms in these patients, it is assumed that their peanut-related symptoms were due to cross-reactive IgE initially raised against pea allergens. Remarkably, patients 1 and 2 were raised in an agricultural environment, where different legumes were cultivated. Possibly their sensitization to pea is the result of early and frequent contact with legumes (skin contact, inhalation, and ingestion) during different stages of ripening. Although early sensitization might have occurred, clinical symptoms did not always develop at a young age. A similar situation might be the case in Mediterranean countries, where lentils and chickpeas are widely consumed and have been described to be more frequently involved in food allergy compared with in Northern Europe. 9 Immunoblotting and IgE inhibition assays were used to identify the involved allergens and to study cross-reactivity in the described patients. Vicilin-like proteins in pea and peanut (Ara h 1) were identified as underlying allergens causing cross-reactivity. Although band patterns of pea vicilin and Ara h 1 differ substantially on SDS-PAGE and immunobloting, the proteins are homologous to a large extent. 19 Pea vicilin consists of a number of polypeptides that all originate from a single precursor protein of 50 kd after posttranslational proteolysis. 28 The posttranslational proteolysis of Ara h 1 is less extensive, resulting in a single band on SDS-PAGE. 25 The observation that IgE binding to Ara h 1 could be inhibited by pea vicilin but IgE binding to pea vicilin could not be inhibited by Ara h 1 suggests that pea acts as the sensitizing agent in these patients, as is also reflected in the course of symptom development in these patients. Two patients also reported symptoms to other legumes. These findings suggest that sensitization to pea FIG 2. IgE ELISA inhibition studies with purified pea vicilin and Ara h 1. Plates were coated with purified pea vicilin (A) or Ara h 1 (B) and, after blocking, incubated with diluted serum from patient 3 that was preincubated with various concentrations of pea vicilin (squares) or Ara h 1 (circles). TABLE II. Concentrations of inhibitors necessary to inhibit initial IgE binding by 50% Ara h 1 IgE interaction Vicilin-IgE interaction IC 50 for IC 50 for IC 50 for IC 50 for Patient Ara h 1 vicilin Ara h 1 vicilin no. (µg/ml) (µg/ml) (µg/ml) (µg/ml) 1 18 <0.01 > > > > IC 50, Inhibitory concentration of 50%. vicilin induces broad cross-reactivity among members of the legume family, which is, at least partly, clinically significant. Burks et al 19 previously described a homologous sequence of 60% to 65% among vicilin-like proteins in peanut (Ara h 1) and pea and speculated that the extensive serologic cross-reactivity among members of the legume family is partly due to IgE directed against vicilin-like proteins. However, patients described in earlier studies usually had adverse reactions to only one food of the legume family These studies mainly concerned persons with peanut allergy. Other studies (from Mediterranean countries) concerned patients allergic to lupine, lentil, or chickpea, and they did mention adverse reactions to other legumes Therefore perhaps the sensitizing legume is of importance in determining clinically significant cross-reactivity among legumes.

5 424 Wensing et al J ALLERGY CLIN IMMUNOL FEBRUARY 2003 The 3 patients described in this study were sensitized to Ara h 1. Ara h 1, identified as a major peanut allergen in 1991, is a 64.5-kd glycoprotein that belongs to the vicilin family of seed storage proteins. 24 Recombinant Ara h 1 was produced in There is some diversity in observed prevalences of Ara h 1 recognition in patients with peanut allergy, varying between 35% and 100%. 24,25,30,31 It is unclear whether these differences can be explained by the use of purified versus recombinant Ara h 1 or by differences in patient characteristics. If peanut sensitization can be explained by cross-reactive IgE in some patients, geographic differences in allergen recognition patterns can be expected as a result of differences in consumption patterns or other environmental factors. The potency of Ara h 1 to inhibit the IgE Ara h 1 interaction on Western blotting is poor in the case of all 3 patients because 150 to 500 µg/ml Ara h 1 is required to obtain partial inhibition. A similar inhibition experiment (not shown) with serum from a patient with peanut allergy who was not sensitized to pea demonstrated that the IgE Ara h 1 interaction was partially inhibited with 1 µg/ml Ara h 1 and completely inhibited with 3 µg/ml Ara h 1. These concentrations are in the same range as needed for vicilin to obtain partial (2 µg/ml) or complete (10 µg/ml) inhibition. The fact that this concentration is approximately 100 times lower than that observed for the presently investigated patients is remarkable and can be explained by the fact that these patients are sensitized to peanut through cross-reactive IgE. Nevertheless, the patients have clear peanut-related symptoms, illustrating biologic activity of this IgE Ara h 1 interaction. Perhaps the less severe reactions described by the patients after ingestion of peanut compared with pea-related symptoms is explained by this different IgE Ara h 1 interaction. Two patients, however, mentioned worsening of their peanutrelated symptoms, and therefore an increase in the severity of peanut-related symptoms over time is not unthinkable. In summary, 3 patients with a history of to pea who had peanut-related symptoms were described. The peanut-related symptoms could be explained by means of cross-reactive IgE initially directed against pea. The molecular basis for this cross-reactivity pattern was formed by vicilin homologues in pea and peanut (Ara h 1). We thank W. J. Koers for his help in recruiting patients with pea allergy and R. Vlooswijk and R. van Biert for their assistance in Western blot and ELISA experiments. REFERENCES 1. Niestijl-Jansen J, Kardinaal A, Huijbers G, Vlieg-Boerstra B, Martens B, Ockhuizen T. 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