# Sunflower seed allergy: Identification of novel 2S-albumins as potential marker allergens

J. Achour, M. Reche, T. Valbuena, C. Chapuis, B. Guillon, O. Galet, K.

Adel-Patient, H. Bernard, Stéphane Hazebrouck

► To cite this version:

J. Achour, M. Reche, T. Valbuena, C. Chapuis, B. Guillon, et al. Sunflower seed allergy: Identification of novel 2S-albumins as potential marker allergens. Allergy, inPress, 10.1111/all.16124. hal-04564636

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1

# Sunflower seed allergy: identification of novel 2S-albumins as potential marker allergens

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*This is the peer reviewed version of the following article:*

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7 Achour, J., Reche, M., Valbuena, T., Chapuis, C., Guillon, B., Galet, O., Adel-Patient, K., Bernard, H. and Hazebrouck, S. (2024), Sunflower seed allergy: Identification of novel 2S-albumins as potential marker allergens. Allergy, 79: 2273-2276. https://doi.org/10.1111/all.16124

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12 This article may be used for non-commercial purposes in accordance with Wiley Terms

13 and Conditions for Use of Self-Archived Versions.
# Sunflower seed allergy: identification of novel 2S-albumins as potential marker allergens

To the editor,

The sensitization prevalence to sunflower seed (SFS) protein is about 2% in European countries.¹ In Spain, around 20% of subjects sensitized to SFS develop clinical symptoms and half of them experienced moderate to severe anaphylaxis.²,³ Only Hel a 3, a non-specific lipid-transfer protein (LTP), is currently recognized as food allergen. However, SFS contain several seed-storage 2S-albumins (SESA) whose allergenicity needs to be evaluated.⁴

We purified the most abundant SESAs: SESA2-1 (Swiss-Prot number A0A161CKQ7, 13.6 kDa), SESA20-2 (A0A161CT49, 15.1 kDa) and SFA-8 (P23110, 12.1 kDa), now indexed in the WHO-IUIS database as Hel a 15, 16 and 17, respectively.⁵ We investigated then the IgE sensitization profile of 14 Spanish patients allergic to SFS (Table 1 and S1). Food allergy was diagnosed by compatible history together with specific IgE (slgE) levels to SFS ranging from 0.4 to 100 kUA/L. Concomitant allergy to tree nuts/peanut (n=12/14, 86%) and/or fruits (n=8/14, 57%) was observed. Sera from 13 patients allergic to nuts and/or fruits, but tolerant to SFS, were included as controls. Of note, percentage of children (< 15 year) in allergic vs control groups was significantly different (3/14 (21%) vs 9/13 (69%), p=0.021 respectively, see supporting information).

First, slgE levels to SFS were quantified by indirect ELISA. However, our assay did not detect significant IgE-binding to any component when slgE levels to SFS were below 0.8 kUA/L (Table S1).

We performed then another immunoassay using immobilized anti-IgE monoclonal antibody to capture IgE antibodies and labeled SFS proteins to detect IgE-binding (see supporting information).

Using this approach, 86% of patients (12/14) were positive to Hel a 15, 79% (11/14) to Hel a 16, and 50% (7/14) to Hel a 17 (Table S2). Most patients allergic to fruits were also sensitized to Hel a 3 (7/8, 88%). The slgE levels to Hel a 3, 15 and 16, but not to Hel a 17, were significantly higher in sera from
39 SFS-allergic patients compared with SFS-tolerant patients (Figure 1A and S1). Moreover, slgE levels to Hel a 15 were higher in patients with moderate (grade 2) or severe (grade 3) symptoms than in patients with mild (grade 1) symptoms (p=0.034, Figure 1B). Although slgE levels to Hel a 15 correlated with those to Hel a 16 (p=0.8274, p=0.0002), but not to Hel a 17 (Fig. S2), the median level of slgE to Hel a 16 was not significantly higher in moderate/severe patients, probably because of our limited number of patients.

45 IgE cross-reactivity was then evidenced among SESAs. For sera 1 and 12, Hel a 15 and Hel a 16 inhibited substantially the IgE-binding to each other and to Hel a 17, even more efficiently than Hel a 17 itself (Fig. 1). Thus, IgE-binding to Hel a 17 was mostly due to cross-reactive IgE primarily directed against other SESAs, as previously observed.⁵ Accordingly, Hel a 15 and 16 were more potent than Hel a 17 in triggering mediator (β-hexosaminidase) release by humanized rat basophilic leukemia (RBL) SX-38 cells (Fig. 1F). Of note, purified peanut 2S-albumins Ara h 2 and Ara h 6 could not inhibit IgE-binding to any SESA (data not shown) but IgE-cross-reactivity with other 2S-albumins from tree-nuts should definitively be investigated (Fig. S3).

53 In conclusion, our study revealed the clinical relevance of the now officially recognized SESAs in SFS allergy. Moreover, IgE sensitization to Hel a 15 and Hel a 16 appeared as a potential marker of clinical severity, which needs to be confirmed in larger cohorts. Our results provide new insights to improve SFS allergy diagnosis. This work focused on LTP and 2S-albumins but allergenicity of helianthinins and oleosins should be also evaluated.⁶

59 **J. Achour**¹, **M. Reche**², **T. Valbuena**², **C. Chapuis**¹, **B. Guillon**¹, **O. Galet**³, **K. Adel-Patient**¹, **H. Bernard**¹,* and **S. Hazebrouck**¹,*

61 ¹Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé, Gif-sur-Yvette, France

63 ²Hospital Universitario Infanta Sofía, San Sebastián de los Reyes, Madrid, Espagne
64 ³Avril SCA 11/13, rue de Monceau, Paris, France

65 * Hervé Bernard and Stéphane Hazebrouck share senior authorship

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67 **Corresponding author:** Stéphane Hazebrouck, UMR CEA INRAE DMTS, CEA de Saclay, bât. 136, RTE DP

68 36, 91191 Gif-sur-Yvette cedex, France

69 Email: stephane.hazebrouck@cea.fr

70

71 **Acknowledgement/funding**

72 We thank Professors Christian Radauer and Richard Goodman, Chairmen of the Executive Committee
73 of the WHO/IUIS Allergen Nomenclature Sub-committee, and the committee members for evaluating
74 and approving the new allergens reported in this work. This project received financial support from
75 SOFIPROTEOL as part of FASO PRODIAL project. JA, SH and KAP participated to the COST Action FA1402
76 – IMPARAS (Improving Allergy Risk Assessment Strategy for New Food Proteins). We thank Victoria
77 Pineau for management of the PRODIAL project.

78

79 **Author contributions**

80 HB, MR, KAP and SH conceptualized the study. MR and TV performed clinical investigations and
81 provided sera. JA, CC, BG, HB and SH performed the experiments. OG supervised the PRODIAL project.
82 HB and SH supervised the study. JA, HB and SH wrote the manuscript. All authors reviewed and
83 approved the final version of the manuscript.

84

85 **Conflict of interest**

86 The authors declare that they have no known competing financial interests or personal relationships
87 that could have appeared to influence the work reported in this manuscript.

88
<table><caption>Table 1: Clinical features from allergic patients and sIgE levels to sunflower seed.</caption><thead><tr><th>Serum</th><th>Age, yr.</th><th>Sex</th><th>SFS allergy</th><th>Symptoms to SFS</th><th>Grade</th><th>Other food allergies</th><th>sIgE to SFS (KUA/L)</th><th>Total IgE (UI/mL)</th></tr></thead><tbody><tr><td>1</td><td>58</td><td>M</td><td>Yes</td><td>Dyspnea, chest tightness</td><td>3</td><td>Almond, cashew, hazelnut, walnut, pistachio, sesame</td><td>39.5</td><td>875.8</td></tr><tr><td>2</td><td>68</td><td>M</td><td>Yes</td><td>Pruritus, generalized urticaria, itching and pharyngeal discomfort, aphonia</td><td>3</td><td>-</td><td>0.4</td><td>26.3</td></tr><tr><td>3</td><td>40</td><td>M</td><td>Yes</td><td>Generalized urticaria, itching and pharyngeal discomfort</td><td>3</td><td>Almond, hazelnut, peanut, walnut, peach</td><td>5.6</td><td>196.6</td></tr><tr><td>4</td><td>41</td><td>M</td><td>Yes</td><td>Oral pruritus</td><td>1</td><td>Peanut, chesnut, walnut, peach, kiwi</td><td>0.7</td><td>147.6</td></tr><tr><td>5</td><td>33</td><td>F</td><td>Yes</td><td>Hypotension, urticaria, angioedema, dyspnea, dizziness</td><td>3</td><td>Mustard</td><td>0.6</td><td>23.9</td></tr><tr><td>6</td><td>43</td><td>F</td><td>Yes</td><td>Urticaria and facial angioedema</td><td>2</td><td>Almond</td><td>0.6</td><td>50.2</td></tr><tr><td>7</td><td>29</td><td>M</td><td>Yes</td><td>Mild chest tightness</td><td>3</td><td>Almond, hazelnut, peanut, chestnut, peach, kiwi</td><td>1.3</td><td>55.1</td></tr><tr><td>8</td><td>6</td><td>M</td><td>Yes</td><td>Facial urticaria</td><td>1</td><td>Almond, cashew, hazelnut, peanut, chesnut, walnut, pistachio, sesame, kiwi</td><td>27.2</td><td>> 5246</td></tr><tr><td>9</td><td>46</td><td>M</td><td>Yes</td><td>Oral pruritus</td><td>1</td><td>Walnut, sesame</td><td>0.8</td><td>7.9</td></tr><tr><td>10</td><td>29</td><td>M</td><td>Yes</td><td>Dizziness, itching and pharyngeal discomfort</td><td>3</td><td>Almond, hazelnut, peanut, walnut</td><td>8.9</td><td>749.8</td></tr><tr><td>11</td><td>15</td><td>F</td><td>Yes</td><td>Pruritus, mild urticaria, rhinitis, itching</td><td>2</td><td>Walnut, hazelnut, peanut, chesnut, peach</td><td>11.4</td><td>1402.3</td></tr><tr><td>12</td><td>14</td><td>F</td><td>Yes</td><td>Cough, dyspnea, wheezing, aphonia, vomiting</td><td>3</td><td>Cashew, pistachio</td><td>100</td><td>608.9</td></tr><tr><td>13</td><td>34</td><td>F</td><td>Yes</td><td>Oral pruritus</td><td>1</td><td>Hazelnut, peanut, lettuce, peach</td><td>0.6</td><td>111.8</td></tr><tr><td>14</td><td>10</td><td>F</td><td>Yes</td><td>Generalized urticaria</td><td>2</td><td>Peanut, peach, kiwi</td><td>9.5</td><td>1079.6</td></tr><tr><td>15</td><td>14</td><td>F</td><td>No</td><td>-</td><td>-</td><td>Almond, cashew, hazelnut, peanut, walnut, pistachio</td><td>7.3</td><td>1571.1</td></tr><tr><td>16</td><td>36</td><td>F</td><td>No</td><td>-</td><td>-</td><td>Peach</td><td>0</td><td>15.2</td></tr><tr><td>17</td><td>6</td><td>F</td><td>No</td><td>-</td><td>-</td><td>Cashew, walnut, pistachio, kiwi</td><td>0</td><td>13.7</td></tr><tr><td>18</td><td>12</td><td>F</td><td>No</td><td>-</td><td>-</td><td>Hazelnut</td><td>0</td><td>81.9</td></tr><tr><td>19</td><td>11</td><td>F</td><td>No</td><td>-</td><td>-</td><td>Hazelnut, peanut, walnut, peach</td><td>0.5</td><td>628.3</td></tr><tr><td>20</td><td>9</td><td>M</td><td>No</td><td>-</td><td>-</td><td>Almond, walnut, hazelnut</td><td>4.3</td><td>3413.7</td></tr><tr><td>21</td><td>15</td><td>M</td><td>No</td><td>-</td><td>-</td><td>Walnut, pistachio, cashew</td><td>0</td><td>261.4</td></tr><tr><td>22</td><td>8</td><td>F</td><td>No</td><td>-</td><td>-</td><td>Hazelnut, peanut, walnut, pine nut</td><td>0</td><td>1523.4</td></tr><tr><td>23</td><td>6</td><td>M</td><td>No</td><td>-</td><td>-</td><td>Almond, cashew, peanut, walnut, pistachio</td><td>0</td><td>579.0</td></tr><tr><td>24</td><td>45</td><td>M</td><td>No</td><td>-</td><td>-</td><td>walnut, mustard</td><td>0.35</td><td>850.0</td></tr><tr><td>25</td><td>11</td><td>F</td><td>No</td><td>-</td><td>-</td><td>Walnut, hazelnut, cashew, kiwi</td><td>nd</td><td>702.2</td></tr><tr><td>26</td><td>5</td><td>F</td><td>No</td><td>-</td><td>-</td><td>Peach, peanut</td><td>0</td><td>419.0</td></tr><tr><td>27</td><td>34</td><td>F</td><td>No</td><td>-</td><td>-</td><td>Peach</td><td>0</td><td>597.6</td></tr></tbody></table>

SFS, sunflower seed; M, male; F, female; Grade 1, mild symptoms; Grade 2, moderate symptoms; Grade 3, severe symptoms; nd, not done.
91 REFERENCES

92 1. Burney P, Summers C, Chinn S, et al. Prevalence and distribution of sensitization to foods in the European Community Respiratory Health Survey: a EuroPrevall analysis. *Allergy*. 2010;65(9):1182-1188.

95 2. Guerrero AA, Macias ML, Gomez F, et al. Subjects Sensitized to Sunflower Seed (Helianthus annuus) Are Tolerant in a High Proportion of Cases. *Journal of Allergy and Clinical Immunology*. 2013;131(2, Supplement):AB87.

98 3. Galleani C, Dieguez MC, Crespo JF. Sunflower Seed Allergy: A Case Series. *Journal of Allergy and Clinical Immunology*. 2021;147(2, Supplement):AB94.

100 4. Franke B, Colgrave ML, Mylne JS, et al. Mature forms of the major seed storage albumins in sunflower: A mass spectrometric approach. *J Proteomics*. 2016;147:177-186.

102 5. Achour J, Guinot M, Guillon B, et al. Sensitization Potency of Sunflower Seed Protein in a Mouse Model: Identification of 2S-Albumins More Allergenic Than SFA-8. *Mol Nutr Food Res.* 2021;65(18):e2100369.

105 6. Barbarroja-Escudero J, Sanchez-Gonzalez MJ, Pineda F, et al. Identification of Lipoproteins From Sunflower Seeds in 2 Monosensitized Anaphylaxis Patients. *J Investig Allergol Clin Immunol*. 2018;28(5):334-336.

108

109
110 **Legends**

111

112 **Figure 1:** Specific IgE levels to SFS (ImmunoCAP, kUA/L) and to individual purified allergens (AU<sub>414nm</sub>) in sera from SFS-allergic patients vs SFS-tolerant patients (A) and in sera from SFS-allergic patients with mild vs moderate/severe symptoms (B). Dotted lines indicate the limit of detection for each allergen and absorbances below these limits were set at 1 AU<sub>414nm</sub>.

116 Medians with interquartile range are shown. Data were analyzed using the nonparametric Mann-Whitney test (\#p < .1, *p<.05, **p<.01, ***p<.001). Competitive inhibition of IgE-binding to Hel a 15 (C), Hel a 16 (D) and Hel a 17 (E). Competitive inhibitions were performed with sera from patients 1 and 12 who were highly sensitized to the 3 different SESAs. Mediator release assay with RBL SX-38 cells passively sensitized with immunopurified total IgE antibodies from sera 1 and 15 (F).

122
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /><figcaption>Figure 1_Achour et al.</figcaption></figure>
Methods

**Patients:** Sera were collected at the Allergology Department of Hospital Universitario Infanta Sofía, in San Sebastián de los Reyes (Madrid, Spain) from 27 patients, aged 5 to 68 (Table 1). Among them, 14 patients were allergic to SFS protein, with SFS-specific IgE > 0.35 kU/L (ImmunoCAP k84, Thermo Fisher Scientific). Serum controls were from 13 patients tolerant to SFS protein but allergic to nuts and/or fruits. Food allergies were diagnosed by compatible history together with proven food sensitization (positive SPT or IgE) and in some patients by oral food challenge (OFC, Table S1). Symptom's severity was graded by following the classification from Muraro et al, 2018.¹ The project was approved by the Research Committee of the Hospital Universitario Infanta Sofía and written informed consent was obtained from each patient or parents of the patient.

The study was designed to determine the IgE sensitization profile of patients allergic to SFS and to identify major SFS allergens. When comparing allergic vs control groups, percentage of children (< 15 yr) was significantly different (3/14 (21%) vs 9/13 (69%), p=0.021 respectively) but not that of male (8/14 (57%) vs 4/13 (31%), p=0.16, respectively). Stratified analyses are presented for children and adults (Fig. S1). Results are comparable to those observed without stratification except for slgE levels to Hel a 3 that are not significantly different in children (Fig. S1A). Among patients allergic to SFS, no significant differences of age and sex were evidenced between the mild and the moderate/severe groups (p>0.99 and p=0.30, respectively).

**Purified components of SFS proteins:** The 2S-albumins Hel a 15/SESA2-1 (Swiss-Prot accession number A0A161CKQ7), Hel a 16/SESA20-2 (A0A161CT49) and Hel a 17/SFA-8 (P23110) and the LTP Hel a 3 (P82007) were purified from a SFS cold press meal obtained after oil extraction (OLEAD, Pessac, France).² Soluble SFS proteins were first separated by 40% ammonium sulfate precipitation. After centrifugation (8000 x g, 30 min, 4 °C), the supernatant (S40) was dialyzed against 20 mM sodium phosphate pH 7.4 buffer (3.5 kDa cut-off). The S40 fraction was filtered (Millex® Syringe filters, 0.22 µm, PES, Merck-Millipore) and submitted to a cation exchange chromatography using a Source 30S column (HR16/10, GE Healthcare), equilibrated with Buffer A (20 mM sodium phosphate pH 7.4). Elution was performed with the following gradient: 0-10 min buffer A and a 0-90 min 0-50% of Buffer B (Buffer A + 1 M NaCl) at a flow rate of 4 mL/min. Collected fractions were further submitted to RP-HPLC using a Vydac C4 column and an AKTA purifier system (GE Healthcare). Elution was performed using a linear gradient from 15% to 60% of solvent B (acetonitrile/ trifluoroacetic acid (TFA) 0.04%) in solvent A (H2O/ TFA 0.1%) at a flow rate of 2 mL/min. Collected fractions were freeze-dried before characterization. Purified proteins were then characterized by mass spectrometry using an Ultraflex MALDI TOF/TOF apparatus (Bruker Daltonics, GmbH). Proteomic analyses were performed following in-solution digestion procedures (Pierce Trypsin Protease™, MS Grade, Thermo Fisher Scientific). Mass spectra were analyzed by using Peptide Analysis® software (Bruker Daltonics, GmbH), and protein identification was carried out by using Peptide Mass (ExPASy server) and a custom-built database. Biotinylation of purified and recombinant SFS proteins was performed with EZ-link® NHS-PEG4-biotin according to manufacturer's instructions (Thermo Fisher Scientific). The protein:biotin molar ratio was at least 1:5 and was adjusted by considering the number of primary amines in each biotinylated protein. The level of labeling was followed by mass spectrometry analysis. Biotinylation was stopped by addition of 1 M Tris buffer.

**Indirect ELISA:** Specific IgE (slgE) levels were first determined by indirect ELISA.³,⁴ Microtiter plates were coated with purified allergens (2.5 µg/mL) and saturated with EIA buffer (0.1 M phosphate buffer, 0.1% BSA, 0.15 M NaCl, 0.01% sodium azide, pH 7.4). After overnight (ON) incubation with sera diluted 1:5 and 1:20, plates were washed and IgE-binding was revealed by addition of a mouse anti-human IgE monoclonal antibody (mAb, clone BS17) labeled with acetylcholinesterase (AchE, 2 Ellman Unit (EU)/mL).⁵ AchE activity was revealed after addition of Ellman's reagent and absorbance was measured at 414 nm.

**IgE-capture ELISA:** Specific IgE levels were also evaluated by measuring the binding of biotinylated allergens to IgE antibodies captured by a mAb immobilized on the solid phase.⁶,⁷ Briefly, mouse anti-human IgE mAb (Clone LE27) was adsorbed on microtiter plates (2.5 µg/mL).⁵ After ON incubation with diluted sera (1:5), plates were washed and biotinylated SFS proteins were added (0.05 nmol/mL) for 4h at room temperature (RT). After washing, AChE-labeled neutravidin was added before revelation with Ellman's reagent. Results were expressed in Absorbance Unit (AU₄₁₄nm). The limit of detection was set at Mean<sub>blank</sub> + at least 3xSD<sub>blank</sub> after measuring 8 replicates obtained with EIA buffer. For competitive inhibition of IgE-binding, after ON incubation with diluted sera (1:5 to 1:100), 25 µL of inhibitors (i.e. increasing concentration of unlabeled SFS protein) were mixed with 25 µL of biotinylated SFS protein (0.05 nmol/mL), and incubated for 4h at RT. IgE-binding was revealed as described above. Results were expressed as B/B₀, B₀ and B representing the amount of labeled SFS protein bound to immobilize IgE antibodies in the absence or presence of a known concentration of inhibitor, respectively.

**Mediator release assay:** Degranulation assays were performed with rat basophilic leukemia (RBL) SX-38 cells.⁸ Cells were passively sensitized with total IgE antibodies from single sera, affinity purified by using anti-IgE mAb (equimolar mix of LE27 and BS17 clones, 1 mg/mL), coupled to CNBr-activated SepharoseTM 4B (GE healthcare, Uppsala, Sweden).⁸ Mediator release was induced by incubation with different SFS proteins and was measured by assaying the β-hexosaminidase activity in culture supernatants. Results were expressed as percentage of the reference release, determined for each serum and induced with anti-human IgE (LE27 clone; 100 ng/mL).

**Statistical analysis:** Data on slgE levels were analyzed with the nonparametric Mann-Whitney test. Fisher's exact test was applied to assess significant differences between the percentages of children (<15 yr) and adults (≥15 yr), and of
females and males. Correlation analyses were performed with the nonparametric Spearman test. Analyses were
performed by using GraphPad Prism 8.3.0 software (La Jolla, Calif). P < .05 was considered statistically significant.

1. Muraro A, Fernandez-Rivas M, Beyer K, et al. The urgent need for a harmonized severity scoring system for acute allergic reactions. *Allergy*. 2018;73(9):1792-1800.

2. Achour J, Guinot M, Guillon B, et al. Sensitization Potency of Sunflower Seed Protein in a Mouse Model: Identification of 2S-Albumins More Allergenic Than SFA-8. *Mol Nutr Food Res.* 2021;65(18):e2100369.

3. Bernard H, Paty E, Mondoulet L, et al. Serological characteristics of peanut allergy in children. *Allergy*. 2003;58(12):1285-1292.

4. Hazebrouck S, Guillon B, Paty E, et al. Variable IgE cross-reactivity between peanut 2S-albumins: The case for measuring IgE to both Ara h 2 and Ara h 6. *Clin Exp Allergy*. 2019;49(8):1107-1115.

5. Grassi J, Didierlaurent A, Stadler BM. Quantitative determination of total and specific human IgE with the use of monoclonal antibodies. *J Allergy Clin Immunol*. 1986;77(6):808-822.

6. Bernard H, Drumare MF, Guillon B, et al. Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays. *Anal Bioanal Chem*. 2009;395(1):139-146.

7. Hazebrouck S, Patil SU, Guillon B, et al. Immunodominant conformational and linear IgE epitopes lie in a single segment of Ara h 2. *J Allergy Clin Immunol*. 2022;150(1):131-139.

8. Blanc F, Adel-Patient K, Drumare MF, et al. Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors. *Clin Exp Allergy*. 2009;39(8):1277-1285.
<table><caption>Table S1: Clinical features from allergic patients and sIgE levels to sunflower seed and purified proteins using indirect ELISA</caption><thead><tr><th>Serum</th><th>Age, yr.</th><th>Sex</th><th>SFS allergy</th><th>Grade</th><th>Other food allergies</th><th>Positive OFC</th><th>Negative OFC</th><th>sIgE to SFS (KUA/L)</th><th>Hel a 3 (UI/mL)</th><th>Hel a 15 (UI/mL)</th><th>Hel a 16 (UI/mL)</th><th>Hel a 17 (UI/mL)</th><th>Total IgE (UI/mL)</th></tr></thead><tbody><tr><td>1</td><td>58</td><td>M</td><td>Yes</td><td>3</td><td>Almond, cashew, hazelnut, walnut, pistachio, sesame</td><td></td><td></td><td>39.5</td><td>1.1</td><td>7.7</td><td>7.1</td><td>2.0</td><td>875.8</td></tr><tr><td>2</td><td>68</td><td>M</td><td>Yes</td><td>3</td><td>-</td><td></td><td>Almond, hazelnut</td><td>0.4</td><td>--</td><td>--</td><td>--</td><td>--</td><td>26.3</td></tr><tr><td>3</td><td>40</td><td>M</td><td>Yes</td><td>3</td><td>Almond, hazelnut, peanut, walnut, peach</td><td></td><td></td><td>5.6</td><td>3.6</td><td>--</td><td>--</td><td>--</td><td>196.6</td></tr><tr><td>4</td><td>41</td><td>M</td><td>Yes</td><td>1</td><td>Peanut, chesnut, walnut, peach, kiwi</td><td></td><td></td><td>0.7</td><td>--</td><td>--</td><td>--</td><td>--</td><td>147.6</td></tr><tr><td>5</td><td>33</td><td>F</td><td>Yes</td><td>3</td><td>Mustard</td><td></td><td>Sesame, almond, hazelnut, walnut, peanut</td><td>0.6</td><td>--</td><td>--</td><td>--</td><td>--</td><td>23.9</td></tr><tr><td>6</td><td>43</td><td>F</td><td>Yes</td><td>2</td><td>Almond</td><td></td><td>Hazelnut, peanut</td><td>0.6</td><td>--</td><td>--</td><td>--</td><td>--</td><td>50.2</td></tr><tr><td>7</td><td>29</td><td>M</td><td>Yes</td><td>3</td><td>Almond, hazelnut, peanut, chestnut, peach, kiwi</td><td></td><td></td><td>1.3</td><td>0.4</td><td>0.3</td><td>--</td><td>--</td><td>55.1</td></tr><tr><td>8</td><td>6</td><td>M</td><td>Yes</td><td>1</td><td>Almond, cashew, hazelnut, peanut, chesnut, walnut, pistachio, sesame, kiwi</td><td></td><td></td><td>27.2</td><td>4.9</td><td>0.5</td><td>0.7</td><td>2.1</td><td>&gt;5246</td></tr><tr><td>9</td><td>46</td><td>M</td><td>Yes</td><td>1</td><td>Walnut, sesame</td><td></td><td></td><td>0.8</td><td>--</td><td>--</td><td>--</td><td>--</td><td>7.9</td></tr><tr><td>10</td><td>29</td><td>M</td><td>Yes</td><td>3</td><td>Almond, hazelnut, peanut, walnut</td><td></td><td></td><td>8.9</td><td>0.3</td><td>--</td><td>--</td><td>1.6</td><td>749.8</td></tr><tr><td>11</td><td>15</td><td>F</td><td>Yes</td><td>2</td><td>Walnut, hazelnut, peanut, chesnut, peach</td><td>Sunflower seeds</td><td></td><td>11.4</td><td>5.4</td><td>0.5</td><td>--</td><td>0.3</td><td>1402.3</td></tr><tr><td>12</td><td>14</td><td>F</td><td>Yes</td><td>3</td><td>Cashew, pistachio</td><td></td><td>Peanut, almond, hazelnut, walnut, sesame</td><td>100</td><td>--</td><td>33.6</td><td>41.4</td><td>9.6</td><td>608.9</td></tr><tr><td>13</td><td>34</td><td>F</td><td>Yes</td><td>1</td><td>Hazelnut, peanut, lettuce, peach</td><td></td><td></td><td>0.6</td><td>--</td><td>--</td><td>--</td><td>--</td><td>111.8</td></tr><tr><td>14</td><td>10</td><td>F</td><td>Yes</td><td>2</td><td>Peanut, peach, kiwi</td><td></td><td>Cashew, almond, walnut</td><td>9.5</td><td>3.4</td><td>--</td><td>--</td><td>--</td><td>1079.6</td></tr><tr><td>15</td><td>14</td><td>F</td><td>No</td><td>-</td><td>Almond, cashew, hazelnut, peanut, walnut, pistachio</td><td></td><td>Pine nut, sunflower seeds</td><td>7.3</td><td>0.4</td><td>--</td><td>--</td><td>0.5</td><td>1571.1</td></tr><tr><td>16</td><td>36</td><td>F</td><td>No</td><td>-</td><td>Peach</td><td></td><td></td><td>--</td><td>--</td><td>--</td><td>--</td><td>--</td><td>15.2</td></tr><tr><td>17</td><td>6</td><td>F</td><td>No</td><td>-</td><td>Cashew, walnut, pistachio, kiwi</td><td></td><td>Peanut, sunflower seeds, pine nut</td><td>--</td><td>--</td><td>--</td><td>--</td><td>--</td><td>13.7</td></tr><tr><td>18</td><td>12</td><td>F</td><td>No</td><td>-</td><td>Hazelnut</td><td></td><td>pistachio</td><td>--</td><td>--</td><td>--</td><td>--</td><td>0.3</td><td>81.9</td></tr><tr><td>19</td><td>11</td><td>F</td><td>No</td><td>-</td><td>Hazelnut, peanut, walnut, peach</td><td></td><td></td><td>0.5</td><td>1.0</td><td>--</td><td>--</td><td>--</td><td>628.3</td></tr><tr><td>20</td><td>9</td><td>M</td><td>No</td><td>-</td><td>Almond, walnut, hazelnut</td><td></td><td>Peanut</td><td>4.3</td><td>--</td><td>--</td><td>--</td><td>0.8</td><td>3413.7</td></tr><tr><td>21</td><td>15</td><td>M</td><td>No</td><td>-</td><td>Walnut, pistachio, cashew</td><td></td><td>Sunflower seeds, peanut, pine nut</td><td>--</td><td>--</td><td>--</td><td>--</td><td>--</td><td>261.4</td></tr><tr><td>22</td><td>8</td><td>F</td><td>No</td><td>-</td><td>Hazelnut, peanut, walnut, pine nut</td><td>Hazelnut</td><td></td><td>--</td><td>--</td><td>--</td><td>0.7</td><td>--</td><td>1523.4</td></tr><tr><td>23</td><td>6</td><td>M</td><td>No</td><td>-</td><td>Almond, cashew, peanut, walnut, pistachio</td><td></td><td></td><td>--</td><td>--</td><td>--</td><td>--</td><td>--</td><td>579.0</td></tr><tr><td>24</td><td>45</td><td>M</td><td>No</td><td>-</td><td>walnut, mustard</td><td></td><td></td><td>0.35</td><td>--</td><td>--</td><td>--</td><td>--</td><td>850.0</td></tr><tr><td>25</td><td>11</td><td>F</td><td>No</td><td>-</td><td>Walnut, hazelnut, cashew, kiwi</td><td></td><td></td><td>nd</td><td>--</td><td>0.4</td><td>--</td><td>0.4</td><td>702.2</td></tr><tr><td>26</td><td>5</td><td>F</td><td>No</td><td>-</td><td>Peach, peanut</td><td></td><td></td><td>--</td><td>--</td><td>--</td><td>--</td><td>--</td><td>419.0</td></tr><tr><td>27</td><td>34</td><td>F</td><td>No</td><td>-</td><td>Peach</td><td></td><td></td><td>--</td><td>--</td><td>--</td><td>--</td><td>--</td><td>597.6</td></tr></tbody></table>

SFS, sunflower seed; OFC, oral food challenge; M, male; F, female; Grade 1, mild symptoms; Grade 2, moderate symptoms; Grade 3, severe symptoms; nd, not done
<table><caption>**Table S2:** IgE sensitization to SFS LTP and SESA as evaluated with the IgE capture immunoassay.</caption><thead><tr><th></th><th>Serum</th><th>Hel a 3</th><th>Hel a 15</th><th>Hel a 16</th><th>Hel a 17</th></tr></thead><tbody><tr><td rowspan="14">Patients allergic to sunflower seed</td><td>1</td><td>34</td><td>1189</td><td>1480</td><td>723</td></tr><tr><td>2</td><td>-</td><td>25</td><td>14</td><td>-</td></tr><tr><td>3</td><td>328</td><td>31</td><td>15</td><td>-</td></tr><tr><td>4</td><td>44</td><td>-</td><td>-</td><td>-</td></tr><tr><td>5</td><td>-</td><td>291</td><td>151</td><td>-</td></tr><tr><td>6</td><td>-</td><td>282</td><td>157</td><td>-</td></tr><tr><td>7</td><td>342</td><td>57</td><td>-</td><td>-</td></tr><tr><td>8</td><td>27</td><td>-</td><td>-</td><td>48</td></tr><tr><td>9</td><td>114</td><td>90</td><td>114</td><td>16</td></tr><tr><td>10</td><td>63</td><td>18</td><td>16</td><td>558</td></tr><tr><td>11</td><td>1095</td><td>328</td><td>53</td><td>62</td></tr><tr><td>12</td><td>19</td><td>3513</td><td>3829</td><td>2601</td></tr><tr><td>13</td><td>28</td><td>17</td><td>16</td><td>33</td></tr><tr><td>14</td><td>582</td><td>28</td><td>20</td><td>-</td></tr><tr><td rowspan="14">Patients tolerant to sunflower seed</td><td>15</td><td>50</td><td>-</td><td>17</td><td>77</td></tr><tr><td>16</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>17</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>18</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>19</td><td>43</td><td>-</td><td>-</td><td>-</td></tr><tr><td>20</td><td>-</td><td>-</td><td>-</td><td>112</td></tr><tr><td>21</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>22</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>23</td><td>-</td><td>-</td><td>-</td><td>83</td></tr><tr><td>24</td><td>-</td><td>21</td><td>14</td><td>20</td></tr><tr><td>25</td><td>-</td><td>-</td><td>13</td><td>-</td></tr><tr><td>26</td><td>43</td><td>16</td><td>-</td><td>-</td></tr><tr><td>27</td><td>-</td><td>17</td><td>-</td><td>-</td></tr></tbody><tfoot><tr><td colspan="6">&lt;12 AU<sub>414nm</sub> 13-24 AU<sub>414nm</sub> 25-99 AU<sub>414nm</sub> 100-199 AU<sub>414nm</sub> 200-399 AU<sub>414nm</sub> &gt;400 AU<sub>414nm</sub></td></tr></tfoot></table>

**Fig. S1:** Specific IgE levels to SFS extract (ImmunoCAP, kUA/L) and to individual purified allergens (AU<sub>414nm</sub>) in sera from SFS-allergic vs SFS-tolerant children (A) and adults (B). Data were analyzed using the nonparametric Mann-Whitney test (\*p < .1, \*\*p < .05, \*\*\*p < .01, \*\*\*\*p < .001).

A. Age < 15 yr

<figure><img src="image_4.png" /></figure>

B. Age ≥ 15 yr

<figure><img src="image_5.png" /></figure>