<figure><img src="image_311.png" /><figcaption>FIGURE 178 *In vitro* cross-reactivity between 11S globulins from tree nuts and seeds. Strong cross-reactivity has been shown for hazelnut and walnut (black arrow). Cross-reactivity only confirmed *in vitro* and limited knowledge regarding clinical relevance is indicated with a grey arrow.</figcaption></figure>

is in contrast to (secondary) sensitization to pollen-related PR10-proteins and profilins, which are labile proteins and are associated with mild to moderate (usually oropharyngeal) symptoms.

**Diagnostic value of IgE measurements to seed storage proteins**

Skin prick test or allergen specific IgE to whole extracts will confirm sensitization to the respective food. In order to define the primary allergen source (Figure 179), measurement of specific IgE to components can be performed. Not all seed storage proteins are available for routine testing. In the ImmunoCAP Specific IgE test single and multiplex assays (Thermo Fisher Scientific), the following seed storage proteins are currently available for routine in vitro diagnostics: peanut rAra h 1, rAra h 2, rAra h 3, rAra h 6, soy nGly m 5, nGly m 6, hazelnut nCor a 9, rCor a 14, cashew nut rAna o 2, rAna o 3, walnut rJug r 1, nJug r 2, Brazil nut rBer e 1, sesame nSes i 1 and buckwheat nFag e 2.

Several studies demonstrated that seed storage proteins play an important role in the diagnosis of peanut, tree nut and seed allergy. They can support the diagnosis of food allergy, but may also give an indication on the severity of the food allergy. For peanut, it was shown that IgE to Ara h 2 has a better diagnostic accuracy than IgE to peanut extract. Cut-off values for Ara h 2 were defined with positive predictive values (PPV) of up to 100% for diagnosis of peanut allergy.<sup>1540</sup> However, one should keep in mind that PPVs depend on the population, setting and geographic location. Measurement of IgE to Ara h 1, 3 and 6 appears less useful in the diagnosis of peanut allergy when IgE to Ara h 2 is already confirmed. However, one study showed that measurement of Ara h 6 could be useful, because co-sensitization to Ara h 2 and Ara h 6 appeared to be associated with severe reactions distinguishing severe allergy from mild symptoms. IgE to hazelnut Cor a 9 and 14 was found to be predictive for clinical reactivity to hazelnut and both were associated with severe reactions.<sup>1544</sup> IgE to walnut Jug r 1 was found to be superior to IgE

to walnut extract in the diagnosis of walnut allergy in children but appeared not to have an additional value in the diagnosis of walnut allergy in adults. IgE to cashew nut Ana o 3 was highly predictive for cashew nut allergy and discriminated between allergic and tolerant children better than cashew nut extract specific IgE; a cut off was found with 95% PPV for diagnosing cashew nut allergy.<sup>1544,1556</sup> The diagnostic value of Ses i 1 appeared to be better than IgE to sesame extract, to Ses i 2 and to 7S and 11S globulins of sesame.<sup>1264</sup> In conclusion, IgE to seed storage proteins has generally a high predictive value to diagnose an allergy to the respective food, has a higher diagnostic value than measurement of IgE to whole extracts, and for some foods, IgE to these allergens is associated with severe clinical reactions. Therefore, measurement of IgE to seed storage proteins is a useful tool in the diagnosis of peanut, tree nut and seed allergy.

**Clinical relevance of cross-sensitizations**

Cross-sensitization within and between legumes, tree nuts and seeds exists, but clinical relevance of these cross-sensitizations varies and may differ between different geographic regions. With regard to legumes, cross-sensitization of peanut allergic patients with other legumes occurs frequently but mostly does not demonstrate clinical allergy.<sup>1067,1538,1552</sup> Lupine may be the most clinically relevant peanut cross-reactive legume, showing sensitization rates of 34-88% and clinically manifested allergy in 4-88% of peanut allergic patients. In peanut allergic patients, sensitization to soybean occurs frequently (31-58%), while clinical allergy to soybean only ranged from 3% to 15%.

Conversely, one study showed that 88% of soybean allergic patients were also peanut allergic. Co-allergies to other legumes (e.g., lentil, chickpea, and pea) in peanut allergic patients have been reported but are less common, especially for beans. Between other legumes, a high degree of IgE cross-reactivity was demonstrated among lentils, chickpeas and peas. Food challenges confirmed that
<figure><img src="image_305.png" /><figcaption>**FIGURE 179** Distinction of primary and secondary sensitization for legumes, tree nuts, and seeds with molecular allergy diagnostics in routine care</figcaption></figure>

clinical allergy to all three legumes was frequently found in a Spanish cohort.<sup>1538,1552,1553</sup>

Peanut and tree nut allergy often co-exists in one patient. Although homology between allergenic proteins of these foods has been demonstrated, the co-allergy between peanut and tree nuts is probably not primarily due to cross-reactivity of IgE to peanut and tree nut allergens.<sup>1554</sup> Taxonomically, peanut and tree nuts are from very different plant groups. Patients in whom co-allergy between peanut and tree nuts exists are likely sensitized to peanut and tree nuts independently. Regarding cross-reactivity of PR-10 proteins, one study showed a high correlation between sensitization to Cor a 1 and Ara h 8, which might indicate that cross-reactivity of PR-10 proteins is a major cause of hazelnut/ peanut co-sensitization.<sup>1555</sup>

Regarding co-allergies between tree nuts, The NUT CRACKER study demonstrated that whilst most patients were sensitized to

5-6 tree nuts, over 50% were only allergic to 1-2 tree-nuts. There is an especially high correlation between walnut-pecan and cashew-pistachio allergies. No association between almond and other tree nuts was found in the NUT CRACKER study. Besides cross-reactivity to seed storage proteins, tree nuts can also show cross-reactivity with pollen, resulting in pollen food allergy syndrome, which is related to milder (usually oropharyngeal) symptoms (Figure 179).

In conclusion, it has to be emphasized that IgE cross-reactivity does not mean that there is also clinically relevant co-allergy. Diagnosis of a legume or tree nut allergy does not automatically imply that all legumes or tree nuts have to be avoided. The oral food challenge is still the gold standard to confirm food allergy and to investigate whether a found co-sensitization is relevant or not.

## Management

Patients with a food allergy to legumes, tree nuts or seeds, especially the ones that are sensitized to seed storage proteins
<figure><img src="image_312.png" /><figcaption>**FIGURE 179** Distinction of primary and secondary sensitization for legumes, tree nuts, and seeds with molecular allergy diagnostics in routine care</figcaption></figure>

clinical allergy to all three legumes was frequently found in a Spanish cohort.<sup>1538,1552,1553</sup>

Peanut and tree nut allergy often co-exists in one patient. Although homology between allergenic proteins of these foods has been demonstrated, the co-allergy between peanut and tree nuts is probably not primarily due to cross-reactivity of IgE to peanut and tree nut allergens.<sup>1554</sup> Taxonomically, peanut and tree nuts are from very different plant groups. Patients in whom co-allergy between peanut and tree nuts exists are likely sensitized to peanut and tree nuts independently. Regarding cross-reactivity of PR-10 proteins, one study showed a high correlation between sensitization to Cor a 1 and Ara h 8, which might indicate that cross-reactivity of PR-10 proteins is a major cause of hazelnut/ peanut co-sensitization.<sup>1555</sup>

Regarding co-allergies between tree nuts, The NUT CRACKER study demonstrated that whilst most patients were sensitized to

5-6 tree nuts, over 50% were only allergic to 1-2 tree-nuts. There is an especially high correlation between walnut-pecan and cashew-pistachio allergies. No association between almond and other tree nuts was found in the NUT CRACKER study. Besides cross-reactivity to seed storage proteins, tree nuts can also show cross-reactivity with pollen, resulting in pollen food allergy syndrome, which is related to milder (usually oropharyngeal) symptoms (Figure 179).

In conclusion, it has to be emphasized that IgE cross-reactivity does not mean that there is also clinically relevant co-allergy. Diagnosis of a legume or tree nut allergy does not automatically imply that all legumes or tree nuts have to be avoided. The oral food challenge is still the gold standard to confirm food allergy and to investigate whether a found co-sensitization is relevant or not.

## Management

Patients with a food allergy to legumes, tree nuts or seeds, especially the ones that are sensitized to seed storage proteins
should avoid the offending food. Furthermore, patients should receive a treatment plan in case of accidental allergic reactions. Patients with severe reactions should receive an adrenaline auto-injector, including a training in how and when to use it as well as an allergy pass.

# 5 - Clinical cases

## Case #1

**Clinical history:** A 21-year old man always developed symptoms of tightness of the throat, urticaria, wheezing and dyspnea after ingestion of peanut. The same symptoms developed after ingestion of green pea and lentil. Beans and products with lupine were ingested without symptoms. He avoided soy his whole life, so it is unknown if that caused symptoms. No symptoms of allergic rhinitis.

**Tests with extracts:** Skin prick test positive for peanut and soybean. Specific IgE to peanut > 100 kU/l, soy 11.5 kU/l, green pea 8.8 kU/l, lentil 6.5 kU/l.

**Tests with molecules:** Ara h 2 79.8 kU/l

**Oral food challenges:** Food challenge with soy was positive at a dose of 0.03 gram soy protein with symptoms of tightness of the throat and urticaria.

**Diagnosis:** Primary peanut allergy with co-allergy to soybean, lentil and green pea. No allergy to lupine or beans.

## Case #2

**Clinical history:** A 10-year old girl had symptoms upon ingestion of a nut mix with walnut, hazelnut, almond and cashew nut consisting of oral allergy symptoms, erythema, nausea and dyspnea. She also had allergic rhinitis. She has never eaten nuts before.

**Test with extracts:** Skin prick test was positive for walnut, hazelnut and birch, negative for almond and cashew nut. Specific IgE to walnut was 2.09 kU/l, hazelnut 9.4 kU/l, almond 0.06 kU/l, cashew nut 0.00 kU/l.

**Test with molecules:** Multiplex assay ISAC: Walnut Jug r 1 3.4 ISU, hazelnut Cor a 1.04 8.7 ISU. Negative for hazelnut Cor a 9, negative for Ana o 2.

**Singleplex assay:** Cor a 14 2.00 kU/l, Cor a 1 12.1 kU/l

**Oral food challenges:** Oral food challenge with hazelnut was positive, symptoms consisted of oral allergy symptoms, tightness of the throat, nausea, vomiting and erythema.

Oral food challenge with walnut was also positive, symptoms consisted of oral allergy symptoms, erythema and dyspnea. Almond and cashew nut were introduced at home without any problem.

**Conclusion:** A primary food allergy to hazelnut and walnut, with in addition also a pollen-related food allergy to hazelnut. Because of high correlation between walnut and pecan nut allergy, also pecan nut has to be avoided. No allergy to almond, no allergy to cashew nut.
should avoid the offending food. Furthermore, patients should receive a treatment plan in case of accidental allergic reactions. Patients with severe reactions should receive an adrenaline auto-injector, including a training in how and when to use it as well as an allergy pass.

# 5 - Clinical cases

## Case #1

**Clinical history:** A 21-year old man always developed symptoms of tightness of the throat, urticaria, wheezing and dyspnea after ingestion of peanut. The same symptoms developed after ingestion of green pea and lentil. Beans and products with lupine were ingested without symptoms. He avoided soy his whole life, so it is unknown if that caused symptoms. No symptoms of allergic rhinitis.

**Tests with extracts:** Skin prick test positive for peanut and soybean. Specific IgE to peanut > 100 kU/l, soy 11.5 kU/l, green pea 8.8 kU/l, lentil 6.5 kU/l.

**Tests with molecules:** Ara h 2 79.8 kU/l

**Oral food challenges:** Food challenge with soy was positive at a dose of 0.03 gram soy protein with symptoms of tightness of the throat and urticaria.

**Diagnosis:** Primary peanut allergy with co-allergy to soybean, lentil and green pea. No allergy to lupine or beans.

## Case #2

**Clinical history:** A 10-year old girl had symptoms upon ingestion of a nut mix with walnut, hazelnut, almond and cashew nut consisting of oral allergy symptoms, erythema, nausea and dyspnea. She also had allergic rhinitis. She has never eaten nuts before.

**Test with extracts:** Skin prick test was positive for walnut, hazelnut and birch, negative for almond and cashew nut. Specific IgE to walnut was 2.09 kU/l, hazelnut 9.4 kU/l, almond 0.06 kU/l, cashew nut 0.00 kU/l.

**Test with molecules:** Multiplex assay ISAC: Walnut Jug r 1 3.4 ISU, hazelnut Cor a 1.04 8.7 ISU. Negative for hazelnut Cor a 9, negative for Ana o 2.

**Singleplex assay:** Cor a 14 2.00 kU/l, Cor a 1 12.1 kU/l

**Oral food challenges:** Oral food challenge with hazelnut was positive, symptoms consisted of oral allergy symptoms, tightness of the throat, nausea, vomiting and erythema.

Oral food challenge with walnut was also positive, symptoms consisted of oral allergy symptoms, erythema and dyspnea. Almond and cashew nut were introduced at home without any problem.

**Conclusion:** A primary food allergy to hazelnut and walnut, with in addition also a pollen-related food allergy to hazelnut. Because of high correlation between walnut and pecan nut allergy, also pecan nut has to be avoided. No allergy to almond, no allergy to cashew nut.
# C09 - Gibberellin-regulated proteins

Pascal Poncet, Tomona Iizuka, Hélène Sénéchal, Enrico Scala

## Highlights

* Gibberellin-regulated proteins (GRPs) are small, cationic, nonglycosylated monomeric proteins with anti-microbial activity, present in plant foods and pollen.

* GRPs are resistant to heat and proteolysis.

* GRPs are cross-reactive and involved in Pollen Food Allergy Syndromes.

* Main fruits involved: peach and citrus but also apricot, cherry, or pomegranate.

* Cupressaceae is, up to now, the only tree family shown to express allergenic pollen GRP.

* GRPs may induce severe systemic reaction with or without cofactors.

## 1 – The protein

The very first Gibberellin-Regulated Protein (GRP) allergen was described in 2013 in peach (Prunus persica) and was named Pru p 7 (formerly peamaclein).⁵⁵⁹ The sensitization was reported in peach allergic patients negative for the other allergens known in peach, especially the nsLTP Pru p 3 that shares some characteristics with Pru p 7, i.e., low molecular weight (MW) and basic isoelectric point (pI). The characterization was refined and confirmed in 2014.¹⁵⁶¹ Pru p 7 is a nonglycosylated, cationic, monomeric protein with an MW around 7-8 kDa and a pI around 9. It belongs to the cysteine-rich plant antimicrobial peptide families that are involved in plant growth and resistance to bacteria, viruses, or other microorganisms that can cause plant disease.¹⁵⁶² Twelve cysteines involved in 6 disulfide bridges confer the protein stability and resistance to heat and proteolysis.

### GRP characteristics

* 6 well-conserved disulfide bridges

* Expressed in pulp and peel of plant food

* Protein present but not synthesized in pollen grain

* Plant defence protein

* The structure displays a cleft likely to bind an unknown ligand

* Pollen/food cross-reactive

## 2 – The family

The family name GRP is now well accepted in the field of allergy although it may not be the most appropriate since the allergens,

with the associated number 7, rather belong to the Snakin/GASA (Gibberellic Acid Stimulated in Arabidopsis) protein family, a sub-family of GRP. Indeed the phytohormone gibberellin regulates very diverse proteins in plants, nonallergenic ones as well as allergenic such as, besides snakin/GASA proteins, superoxide dismutase, β-1,3-glucanase, calmodulin or oleosin.¹⁵⁶³

Gibberellin is a phytohormone produced by all plants, some fungi and bacteria. It corresponds to a family of tetracyclic diterpenic molecules playing a role in plant growth and breaking dormancy.¹⁵⁶⁴ Gibberellin and GRP have an important role in plant development, host defence and redox homeostasis. Consequently, their concentration is strictly regulated and may be different in specific developmental stages. Furthermore, both biotic and abiotic stresses could influence GRP levels.¹⁵⁶⁵ Nowadays gibberellins are widely used in modern agriculture to increase the yield and/or quality of plant food.¹⁵⁶⁶ Numerous plant foods are submitted to an exogenous gibberellin treatment such as grape, cherry, strawberry, pear, tangerine, plum, orange, blueberry, pineapple, tomato, potato, wheat, rice, barley, hop, sunflower, alfalfa (Medicago), chili/red pepper, zucchini, salad, spinach, celery or cotton. By consequence, the utilization of exogenous synthetic gibberellin might affect the concentration of GRPs synthesized in plant foods and even in pollens, therefore influencing also their allergenic potency.

Once produced, GRPs contain a signal peptide of 25 amino-acid that is subsequently removed to obtain the protein mature form of 7 kDa (63 AA). Mature GRPs are structurally characterized by a highly conserved C-terminal region and, as in Pru p 7, by the 12 cysteines at conserved positions. GRPs are water-soluble proteins positively charged at neutral pH with a compact globular conformation, which may result in over-evaluation of its MW depending on the bio- and physicochemical analytical methods used. The protein folding is responsible for conformational epitopes destroyed upon *in vitro* reduction in disulfide bonds.

Snakin-1, the first GRP described in 1999, was isolated from Solanum tuberosum from the potato plant tuber allowing extensive studies on its structure and antimicrobial activity.¹⁵⁶⁵ The three-dimensional structure of snakin-1 was obtained by X-ray crystallography.¹⁵⁶⁷ The folding of the protein comprises three alpha-helices and a cleft likely able to accommodate one or more ligands, as yet undetermined (Figure 180).

Snakin-1 is not yet described as an allergen. After the description of Pru p 7, Pun g 7 a GRP from pomegranate (Punica granatum) was reported¹⁵⁶⁸ as well as Pru m 7, the GRP from Japanese apricot (Prunus mume).¹⁵⁶⁹ In Japan, Japanese apricots are traditionally consumed marinated in salt, they are named umeboshi. More fruits were suspected to contain allergenic GRPs,¹¹³⁰ but convincing data were subsequently obtained only for orange (Citrus sinensis) and sweet cherry (Prunus avium), Cit s 7¹⁵⁷⁰ and Pru av 7, respectively (IUIS/WHO Pru av 7 descriptions: http://www.allergen.org/viewallergen.php?aid=1002). Grapefruit, tangerine and lemon contain cross-reactive GRP with orange (Poncet et al., unpublished results).

A breakthrough was provided by the study of allergenic GRPs when it was demonstrated that an allergen from the Cupressaceae
# C09 - Gibberellin-regulated proteins

Pascal Poncet, Tomona Iizuka, Hélène Sénéchal, Enrico Scala

## Highlights

* Gibberellin-regulated proteins (GRPs) are small, cationic, nonglycosylated monomeric proteins with anti-microbial activity, present in plant foods and pollen.

* GRPs are resistant to heat and proteolysis.

* GRPs are cross-reactive and involved in Pollen Food Allergy Syndromes.

* Main fruits involved: peach and citrus but also apricot, cherry, or pomegranate.

* Cupressaceae is, up to now, the only tree family shown to express allergenic pollen GRP.

* GRPs may induce severe systemic reaction with or without cofactors.

## 1 - The protein

The very first Gibberellin-Regulated Protein (GRP) allergen was described in 2013 in peach (Prunus persica) and was named Pru p 7 (formerly peamaclein).⁵⁵⁹ The sensitization was reported in peach allergic patients negative for the other allergens known in peach, especially the nsLTP Pru p 3 that shares some characteristics with Pru p 7, i.e., low molecular weight (MW) and basic isoelectric point (pI). The characterization was refined and confirmed in 2014.¹⁵⁶¹ Pru p 7 is a nonglycosylated, cationic, monomeric protein with an MW around 7-8 kDa and a pI around 9. It belongs to the cysteine-rich plant antimicrobial peptide families that are involved in plant growth and resistance to bacteria, viruses, or other microorganisms that can cause plant disease.¹⁵⁶² Twelve cysteines involved in 6 disulfide bridges confer the protein stability and resistance to heat and proteolysis.

### GRP characteristics

* 6 well-conserved disulfide bridges

* Expressed in pulp and peel of plant food

* Protein present but not synthesized in pollen grain

* Plant defence protein

* The structure displays a cleft likely to bind an unknown ligand

* Pollen/food cross-reactive

## 2 - The family

The family name GRP is now well accepted in the field of allergy although it may not be the most appropriate since the allergens,

with the associated number 7, rather belong to the Snakin/GASA (Gibberellic Acid Stimulated in Arabidopsis) protein family, a sub-family of GRP. Indeed the phytohormone gibberellin regulates very diverse proteins in plants, nonallergenic ones as well as allergenic such as, besides snakin/GASA proteins, superoxide dismutase, β-1,3-glucanase, calmodulin or oleosin.¹⁵⁶³

Gibberellin is a phytohormone produced by all plants, some fungi and bacteria. It corresponds to a family of tetracyclic diterpenic molecules playing a role in plant growth and breaking dormancy.¹⁵⁶⁴ Gibberellin and GRP have an important role in plant development, host defence and redox homeostasis. Consequently, their concentration is strictly regulated and may be different in specific developmental stages. Furthermore, both biotic and abiotic stresses could influence GRP levels.¹⁵⁶⁵ Nowadays gibberellins are widely used in modern agriculture to increase the yield and/or quality of plant food.¹⁵⁶⁶ Numerous plant foods are submitted to an exogenous gibberellin treatment such as grape, cherry, strawberry, pear, tangerine, plum, orange, blueberry, pineapple, tomato, potato, wheat, rice, barley, hop, sunflower, alfalfa (Medicago), chili/red pepper, zucchini, salad, spinach, celery or cotton. By consequence, the utilization of exogenous synthetic gibberellin might affect the concentration of GRPs synthesized in plant foods and even in pollens, therefore influencing also their allergenic potency.

Once produced, GRPs contain a signal peptide of 25 amino-acid that is subsequently removed to obtain the protein mature form of 7 kDa (63 AA). Mature GRPs are structurally characterized by a highly conserved C-terminal region and, as in Pru p 7, by the 12 cysteines at conserved positions. GRPs are water-soluble proteins positively charged at neutral pH with a compact globular conformation, which may result in over-evaluation of its MW depending on the bio- and physicochemical analytical methods used. The protein folding is responsible for conformational epitopes destroyed upon *in vitro* reduction in disulfide bonds.

Snakin-1, the first GRP described in 1999, was isolated from Solanum tuberosum from the potato plant tuber allowing extensive studies on its structure and antimicrobial activity.¹⁵⁶⁵ The three-dimensional structure of snakin-1 was obtained by X-ray crystallography.¹⁵⁶⁷ The folding of the protein comprises three alpha-helices and a cleft likely able to accommodate one or more ligands, as yet undetermined (Figure 180).

Snakin-1 is not yet described as an allergen. After the description of Pru p 7, Pun g 7 a GRP from pomegranate (Punica granatum) was reported¹⁵⁶⁸ as well as Pru m 7, the GRP from Japanese apricot (Prunus mume).¹⁵⁶⁹ In Japan, Japanese apricots are traditionally consumed marinated in salt, they are named umeboshi. More fruits were suspected to contain allergenic GRPs,¹¹³⁰ but convincing data were subsequently obtained only for orange (Citrus sinensis) and sweet cherry (Prunus avium), Cit s 7¹⁵⁷⁰ and Pru av 7, respectively (IUIS/WHO Pru av 7 descriptions: http://www.allergen.org/viewallergen.php?aid=1002). Grapefruit, tangerine and lemon contain cross-reactive GRP with orange (Poncet et al., unpublished results).

A breakthrough was provided by the study of allergenic GRPs when it was demonstrated that an allergen from the Cupressaceae
<figure><img src="image_306.png" /><figcaption>FIGURE 180 Three-dimensional structure of Snakin-1 (PDB 5E5Q). Ribbon representation with (A) or without surface (B).</figcaption></figure>

pollen first reported in 2010,¹⁵⁷¹ the formerly called BP14, was shown to belong to the GRP protein family.¹⁵⁷² The pollen food associated syndrome (PFAS) between peach or citrus and cypress pollen reported in 2006¹⁵⁷³ and 2015¹⁵⁷⁴ was thus explained by the existence of an IgE cross-reactivity between Pru p 7 or Cit s 7 and the allergen BP14.²³⁰,¹⁵⁷⁵,¹⁵⁷⁶ The gene coding for BP14 was then fully sequenced from common cypress (Cupressus sempervirens) strobili by next-generation sequencing and the protein named Cup s 7 (IUIS/WHO Description of Cup s 7, http://www.allergen.org/viewallergen.php?aid=997). A homologous allergen, Cry j 7, with similar fruit cross-reactivities, was then described in Japanese cedar pollen (Cryptomeria japonica) by studying Japanese patients allergic to Japanese cedar pollen and food.¹⁵⁷⁸ As well the existence of a mountain cedar pollen (Juniperus ashei) GRP, Jun a 7, was confirmed.¹¹⁵⁷ We could expect that other trees from the Cupressaceae family such as the Japanese cypress (Chamaecyparis obtusa) or the bald cypress (Taxodium distichum) also express an allergenic pollen GRP.

Finally, in 2021, an allergenic GRP, Cap a 7, was revealed in bell pepper (Capsicum annuum) by studying a Japanese patient allergic to several GRPs, from bell/chili pepper (Cap a 7), from peach (Pru p 7), orange (Cit s 7) and from Japanese cedar pollen (Cry j 7) demonstrating a clinical relevance of the cross-reactivities between different GRPs (IUIS/WHO Description of Cap a 7, http://www.allergen.org/viewallergen.php?aid=1061).

Up to now, GRPs from only 9 allergenic sources have been described as allergens. Five from fruits, 1 from a vegetable and 3 from tree pollen, all belonging to the Cupressaceae family (Tables 99 and 100).

Cupressaceae GRPs are very similar with more than 90% sequence identity and share more than 60% sequence identity with fruit and vegetables. Similarly, the percentage of sequence identity between fruit GRPs or vegetable GRPs are high and close to each other. Therefore, all GRP should theoretically be cross-reactive (Table 101). However, the cross-reactivity is not always experimentally observed.¹⁵⁷⁸

The relationships between the different taxa and the 3-dimensional modelling of proteins are depicted in (Figure 181). The pollen GRPs are more distant from plant food-derived ones. Snakin-1 from potato and citrus fruits such as grapefruit (Citrus

maxima), tangerine (Citrus reticulata) and lemon (Citrus limone) are depicted on a yellow background because the GRP allergens are not fully characterized. However, GRP cross-reactivities were shown among citrus fruits and a clementine (Citrus clementina) GRP is described in the Uniprot KB database (accession number V4T144) with a 100% sequence identity with orange GRP.

Three-dimensional modelling using potato snakin-1 as a template showed a few structural differences between the various GRPs that could lead to variations in the size of the three epitope regions predicted by the software DiscoTope 2.0 (Figure 181).

## 3 – Clinical relevance

GRPs are found in both, pulp and peel of fruits, in contrast to nsLTPs, which are mainly present in the peel, and to a lower extent in the pulp. However, bell pepper GRP was only found in the pulp (unpublished results). Interestingly GRPs can be present or absent in different fruit cultivars, even in distinct lots belonging to the same cultivar.⁵⁵⁹,¹⁵⁶⁸

Very often the sensitization to fruit GRPs is associated with Cupressaceae pollen allergy. This was observed for Mediterranean cypress in Europe¹⁵⁷⁶,²³¹ as well as for Japanese cedar in Japan.¹⁵⁷⁷ Forty-six per cent of young Japanese patients allergic to Japanese cedar pollen and fruit are sensitized to GRPs. This observation suggests a possible interdependence of both sensitizations. The association might rely not only on the cross-reactivity between Cupressaceae and fruit GRPs but also on a sensitization process involving some specific ligand-protein interactions common between the two allergenic sources that synergise the allergic response towards GRPs. Interestingly, in the case of allergy to GRPs, sensitization to cypress pollen does not necessarily involve Cup a 1, the major allergen of Cupressaceae pollen. It is not known whether sensitization to GRPs from cypress (i.e., Cup s 7 or Cry j 7), in the absence of recognition of Cup a 1, can generate respiratory symptoms or not. At the same time, it is not known whether sensitization to food GRPs necessarily follows a sensitization to pollen (as in the case of PR-10 or Profilin; chapters C01, C02) or can be directly caused by fruits, acting as primary sensitizers (as for nsLTPs, in the Mediterranean area). The main fruits involved are peach and citrus. Pomegranate allergy seems much rarer and the only patient allergic to bell/chili pepper was also allergic to Japanese cedar pollen, peach and citrus (see clinical case #4). GRP cross-reactivities that are immunochemically assessed using recombinant protein may not be clinically relevant. This was observed with snakin-1 able to be bound by IgEs from a cypress/peach allergic patient (Cup s 7+/ Pru p 7+) but unable to activate the patient's basophils in agreement with the tolerance of potato consumption by the patient.²³⁰ Differences in antibody affinity probably play a role. At least two pollen food allergy syndromes were previously described between cypress pollen and peach and/or citrus.¹⁵⁷³,¹⁵⁷⁴ GRPs should be clinically relevant since they can induce ex-vivo basophil activation.²³⁰,²³¹,⁵⁵⁹ GRP allergies were reported to be more common in adolescents
A

<figure><img src="image_313.png" /></figure>

B

<figure><img src="image_314.png" /><figcaption>FIGURE 180 Three-dimensional structure of Snakin-1 (PDB 5E5Q). Ribbon representation with (A) or without surface (B).</figcaption></figure>

pollen first reported in 2010,¹⁵⁷¹ the formerly called BP14, was shown to belong to the GRP protein family.¹⁵⁷² The pollen food associated syndrome (PFAS) between peach or citrus and cypress pollen reported in 2006¹⁵⁷³ and 2015¹⁵⁷⁴ was thus explained by the existence of an IgE cross-reactivity between Pru p 7 or Cit s 7 and the allergen BP14.²³⁰,¹⁵⁷⁵,¹⁵⁷⁶ The gene coding for BP14 was then fully sequenced from common cypress (Cupressus sempervirens) strobili by next-generation sequencing and the protein named Cup s 7 (IUIS/WHO Description of Cup s 7, http://www.allergen.org/viewallergen.php?aid=997). A homologous allergen, Cry j 7, with similar fruit cross-reactivities, was then described in Japanese cedar pollen (Cryptomeria japonica) by studying Japanese patients allergic to Japanese cedar pollen and food.¹⁵⁷⁸ As well the existence of a mountain cedar pollen (Juniperus ashei) GRP, Jun a 7, was confirmed.¹¹⁵⁷ We could expect that other trees from the Cupressaceae family such as the Japanese cypress (Chamaecyparis obtusa) or the bald cypress (Taxodium distichum) also express an allergenic pollen GRP.

Finally, in 2021, an allergenic GRP, Cap a 7, was revealed in bell pepper (Capsicum annuum) by studying a Japanese patient allergic to several GRPs, from bell/chili pepper (Cap a 7), from peach (Pru p 7), orange (Cit s 7) and from Japanese cedar pollen (Cry j 7) demonstrating a clinical relevance of the cross-reactivities between different GRPs (IUIS/WHO Description of Cap a 7, http://www.allergen.org/viewallergen.php?aid=1061).

Up to now, GRPs from only 9 allergenic sources have been described as allergens. Five from fruits, 1 from a vegetable and 3 from tree pollen, all belonging to the Cupressaceae family (Tables 99 and 100).

Cupressaceae GRPs are very similar with more than 90% sequence identity and share more than 60% sequence identity with fruit and vegetables. Similarly, the percentage of sequence identity between fruit GRPs or vegetable GRPs are high and close to each other. Therefore, all GRP should theoretically be cross-reactive (Table 101). However, the cross-reactivity is not always experimentally observed.¹⁵⁷⁸

The relationships between the different taxa and the 3-dimensional modelling of proteins are depicted in (Figure 181). The pollen GRPs are more distant from plant food-derived ones. Snakin-1 from potato and citrus fruits such as grapefruit (Citrus

maxima), tangerine (Citrus reticulata) and lemon (Citrus limone) are depicted on a yellow background because the GRP allergens are not fully characterized. However, GRP cross-reactivities were shown among citrus fruits and a clementine (Citrus clementina) GRP is described in the Uniprot KB database (accession number V4T144) with a 100% sequence identity with orange GRP.

Three-dimensional modelling using potato snakin-1 as a template showed a few structural differences between the various GRPs that could lead to variations in the size of the three epitope regions predicted by the software DiscoTope 2.0 (Figure 181).

## 3 - Clinical relevance

GRPs are found in both, pulp and peel of fruits, in contrast to nsLTPs, which are mainly present in the peel, and to a lower extent in the pulp. However, bell pepper GRP was only found in the pulp (unpublished results). Interestingly GRPs can be present or absent in different fruit cultivars, even in distinct lots belonging to the same cultivar.⁵⁵⁹,¹⁵⁶⁸

Very often the sensitization to fruit GRPs is associated with Cupressaceae pollen allergy. This was observed for Mediterranean cypress in Europe¹⁵⁷⁶,²³¹ as well as for Japanese cedar in Japan.¹⁵⁷⁷ Forty-six per cent of young Japanese patients allergic to Japanese cedar pollen and fruit are sensitized to GRPs. This observation suggests a possible interdependence of both sensitizations. The association might rely not only on the cross-reactivity between Cupressaceae and fruit GRPs but also on a sensitization process involving some specific ligand-protein interactions common between the two allergenic sources that synergise the allergic response towards GRPs. Interestingly, in the case of allergy to GRPs, sensitization to cypress pollen does not necessarily involve Cup a 1, the major allergen of Cupressaceae pollen. It is not known whether sensitization to GRPs from cypress (i.e., Cup s 7 or Cry j 7), in the absence of recognition of Cup a 1, can generate respiratory symptoms or not. At the same time, it is not known whether sensitization to food GRPs necessarily follows a sensitization to pollen (as in the case of PR-10 or Profilin; chapters C01, C02) or can be directly caused by fruits, acting as primary sensitizers (as for nsLTPs, in the Mediterranean area). The main fruits involved are peach and citrus. Pomegranate allergy seems much rarer and the only patient allergic to bell/chili pepper was also allergic to Japanese cedar pollen, peach and citrus (see clinical case #4). GRP cross-reactivities that are immunochemically assessed using recombinant protein may not be clinically relevant. This was observed with snakin-1 able to be bound by IgEs from a cypress/peach allergic patient (Cup s 7+/ Pru p 7+) but unable to activate the patient's basophils in agreement with the tolerance of potato consumption by the patient.²³⁰ Differences in antibody affinity probably play a role. At least two pollen food allergy syndromes were previously described between cypress pollen and peach and/or citrus.¹⁵⁷³,¹⁵⁷⁴ GRPs should be clinically relevant since they can induce ex-vivo basophil activation.²³⁰,²³¹,⁵⁵⁹ GRP allergies were reported to be more common in adolescents
<figure><img src="image_307.png" /></figure>

FIGURE 181 Evolutionary relationships of taxa (phylogenetic tree) and 3D modelling of nine allergenic GRP and the prototype reference GRP snakin-1 from potato. The evolutionary history was inferred using the Neighbor-Joining method<sup>1579</sup> and evolutionary analyses were conducted in MEGA X<sup>1580</sup>. Three-dimensional structure modelling of proteins was calculated using snakin-1 as a template. Three conformational epitopic regions were predicted using the software Disco Tope 2.0. They are coloured in yellow and orange.

extracts showed identical performances, while in the case of labile allergen (Pru p 1 or Pru p 4) reactivities, they consistently gave a negative result. This extract evaluation is therefore an excellent approach to identify reactivity to nsLTP or, possibly Pru p 7, during the first screening of the patient.<sup>1583</sup>

In another multicenter Italian study, 835 cypress pollen hyper-sensitive patients were evaluated with a peach extract containing Pru p 7 by SPT. In peach sensitized individuals, IgE to rPru p 3 was evaluated, and only those scoring negative were further studied for IgE reactivity to rPru p 7 by immunoblot and by an, at that time, "ex-perimental" ImmunoCAP Specific IgE test with rPru p 7. Peach SPT reactivity was found in 163 (19.5%) patients, but 127 (77.9%) were excluded because they were also Pru p 3 reactors. On immunoblot, only 3/18 subjects recognized a band at about 7 kDa. Ten/18 (56%)

were Pru p 7 reactors on ImmunoCAP Specific IgE test. Taken together, Pru p 7 allergy seem to occur quite rarely in Italy (less than 3% among cypress reactors<sup>592</sup>).

GRP sensitization has to be suspected after systemic reactions that could have been associated with well-known cofactors such as physical exercise, NSAID, alcohol, proton pump inhibitors when the fruit has been consumed. Since Cupressaceae pollen allergy is a very frequent association with GRP sensitization, such pollen sensitization should be carefully evaluated even though the association mechanism is, up to now, not well understood. Cypress pollen reactivity, even after skin prick test, in the absence of Cup a 1, polcalcin or CCD IgE recognition may occur in case of GRPs' sensitization. Then, specific IgE against nsLTP is usually negative as well as against profilin. Interestingly slgE against nsLTP or profilin was not reported in the case of cypress pollen allergy. The recombinant Pru p 7-specific IgE test, commercially available in singleplex and multiplex assays, may help in the diagnosis although a positive GRP immunoassay might not be associated with a clinically relevant IgE reactivity. Therefore, a method evaluating the IgE reactivity to the natural GRP may be helpful to confirm the diagnosis, for instance, immunoblot with total extract in nonreducing conditions. To complete the diagnosis an ex-vivo basophil activation test could be performed with total extract and with the recombinant GRP since a positive basophil activation test, in contrast to immunoassays, strongly suggests a potential clinical relevance. Because severe reactions such as anaphylactic shock were reported, an adrenaline autoinjector should be recommended to the patient as well as avoidance of the culprit food in both raw and processed forms when the diagnosis is established. An algorithm is presented in (Figure 182).

## 5 - Clinical cases

### Case 1 (original):

**Clinical History:** Male, Italy, born in 2002. Patient suffering from seasonal allergic rhinitis every year between January and March. He reported three episodes of anaphylactic reaction characterized by hypotension and diffuse urticaria with angioedema during dinner, after the ingestion of (2015) a slice of peeled peach, (2016) pomegranate (2018), and (2020) a few slices of orange. The patient in all cases was brought to the ER, where he received a combination of intramuscular adrenalin and intravenous steroid.

**Allergy testing:** The patient went through a cutaneous allergic evaluation that gave positive results for cypress pollen (10mm x 7mm) and a commercial peach extract containing 30 mg/ml of Pru p 3 (12mm x 9mm). He was then tested for IgE to cypress: 15 kUA/L; peach: 3.5 kUA/L; Pru p 1: <0.1 kUA/L, Pru p 4: <0.1 kUA/L, Pru p 3: <0.1 kUA/L and MUXF3: <0.1 kUA/L. A year later, the patient was further tested, scoring positive for Pru p 7: 14.7 kUA/L.

**Conclusion:** The serology identifies the patient as genuinely sensitized to Pru p 7. The presence of positive results after SPT to peach extract in the absence of PR-10, Profilin, nsLTP or CCD reactivity indicates a strong suspicion for GRP sensitization. Nowadays
<figure><img src="image_315.png" /><figcaption>FIGURE 181 Evolutionary relationships of taxa (phylogenetic tree) and 3D modelling of nine allergenic GRP and the prototype reference GRP snakin-1 from potato. The evolutionary history was inferred using the Neighbor-Joining method<sup>1579</sup> and evolutionary analyses were conducted in MEGA X<sup>1580</sup>. Three-dimensional structure modelling of proteins was calculated using snakin-1 as a template. Three conformational epitopic regions were predicted using the software Disco Tope 2.0. They are coloured in yellow and orange.</figcaption></figure>

extracts showed identical performances, while in the case of labile allergen (Pru p 1 or Pru p 4) reactivities, they consistently gave a negative result. This extract evaluation is therefore an excellent approach to identify reactivity to nsLTP or, possibly Pru p 7, during the first screening of the patient.<sup>1583</sup>

In another multicenter Italian study, 835 cypress pollen hyper-sensitive patients were evaluated with a peach extract containing Pru p 7 by SPT. In peach sensitized individuals, IgE to rPru p 3 was evaluated, and only those scoring negative were further studied for IgE reactivity to rPru p 7 by immunoblot and by an, at that time, "ex-perimental" ImmunoCAP Specific IgE test with rPru p 7. Peach SPT reactivity was found in 163 (19.5%) patients, but 127 (77.9%) were excluded because they were also Pru p 3 reactors. On immunoblot, only 3/18 subjects recognized a band at about 7 kDa. Ten/18 (56%)

were Pru p 7 reactors on ImmunoCAP Specific IgE test. Taken together, Pru p 7 allergy seem to occur quite rarely in Italy (less than 3% among cypress reactors<sup>592</sup>).

GRP sensitization has to be suspected after systemic reactions that could have been associated with well-known cofactors such as physical exercise, NSAID, alcohol, proton pump inhibitors when the fruit has been consumed. Since Cupressaceae pollen allergy is a very frequent association with GRP sensitization, such pollen sensitization should be carefully evaluated even though the association mechanism is, up to now, not well understood. Cypress pollen reactivity, even after skin prick test, in the absence of Cup a 1, polcalcin or CCD IgE recognition may occur in case of GRPs' sensitization. Then, specific IgE against nsLTP is usually negative as well as against profilin. Interestingly slgE against nsLTP or profilin was not reported in the case of cypress pollen allergy. The recombinant Pru p 7-specific IgE test, commercially available in singleplex and multiplex assays, may help in the diagnosis although a positive GRP immunoassay might not be associated with a clinically relevant IgE reactivity. Therefore, a method evaluating the IgE reactivity to the natural GRP may be helpful to confirm the diagnosis, for instance, immunoblot with total extract in nonreducing conditions. To complete the diagnosis an ex-vivo basophil activation test could be performed with total extract and with the recombinant GRP since a positive basophil activation test, in contrast to immunoassays, strongly suggests a potential clinical relevance. Because severe reactions such as anaphylactic shock were reported, an adrenaline autoinjector should be recommended to the patient as well as avoidance of the culprit food in both raw and processed forms when the diagnosis is established. An algorithm is presented in (Figure 182).

## 5 - Clinical cases

### Case 1 (original):

**Clinical History:** Male, Italy, born in 2002. Patient suffering from seasonal allergic rhinitis every year between January and March. He reported three episodes of anaphylactic reaction characterized by hypotension and diffuse urticaria with angioedema during dinner, after the ingestion of (2015) a slice of peeled peach, (2016) pomegranate (2018), and (2020) a few slices of orange. The patient in all cases was brought to the ER, where he received a combination of intramuscular adrenalin and intravenous steroid.

**Allergy testing:** The patient went through a cutaneous allergic evaluation that gave positive results for cypress pollen (10mm x 7mm) and a commercial peach extract containing 30 mg/ml of Pru p 3 (12mm x 9mm). He was then tested for IgE to cypress: 15 kUA/L; peach: 3.5 kUA/L; Pru p 1: <0.1 kUA/L, Pru p 4: <0.1 kUA/L, Pru p 3: <0.1 kUA/L and MUXF3: <0.1 kUA/L. A year later, the patient was further tested, scoring positive for Pru p 7: 14.7 kUA/L.

**Conclusion:** The serology identifies the patient as genuinely sensitized to Pru p 7. The presence of positive results after SPT to peach extract in the absence of PR-10, Profilin, nsLTP or CCD reactivity indicates a strong suspicion for GRP sensitization. Nowadays
<figure><img src="image_308.png" /><figcaption>FIGURE 182 Diagnostic algorithm to assess GRP sensitization</figcaption></figure>

it is possible to in vitro test Pru p 7, and this analysis should always be included in allergy work-out in patients with severe reactions to *Rosaceae*, pomegranate, or citrus fruits.

**Case 2 (original):**

**Clinical History:** Female, Italy, born 1990. The patient has been suffering from seasonal allergic rhinitis between February and March since 2000. After ingestion of a peeled peach and about 30 min running, she had an anaphylactic reaction (low blood pressure, abdominal pain, generalized flushing and swelling, followed by respiratory difficulty due to laryngeal obstruction) and subsequent emergency treatment. Another similar adverse reaction occurred after ingestion of two walnuts associated with moderate physical exercise.

**Allergy testing:** (A) SPT: Environmental allergens: Cypress pollen (*Juniperus a.):* 10mm × 6mm; plane tree (*Platanus a.):* 5mm × 6mm; olive tree (*Olea e.):* 3mm × 2mm; mugwort (*Artemisia v.):* 7mm × 4mm. Food allergens: all negative except walnut (*Juglans r. nut*): 7mm × 5mm and peach (*Prunus p.):* 19mm × 8mm. (B) *In-vitro* testing: [2015] Total IgE 350.3 kU/L, specific IgE to Cypress pollen (*Cupressus a.):* 12.5 kU/L; plane tree (*Platanus a.): 0.66 kU/L; Olive tree (*Olea e.):* 0.12 kU/L; Mugwort (*Artemisia v.):* 2.2 kU/L; Walnut (*Juglans r. nut*) 3.82 kU/L; rPru p 3: 1.79 kU/L.

After 6 years, the patient returned to visit reporting a further reaction after physical exertion (bicycle) performed after ingesting an orange. The patient was studied with a multiplex method, which allowed to highlight, in addition to the already known

reactivity to Cypress (Cry j 1: 2.31 kU/L and Cup a 1: 31.93 kU/L) and nsLTP (Ole e 7: 1.26 kU/L; Cor a 8: 0.94 kU/L; Jug r 3: 0.35 kU/L; Art v 3: 0.52 kU/L; Pru p 3: 2.42 kU/L), also the presence of reactivity to Pru p 7 (8.34 kU/L). Interestingly, the 2015 serum stored in our serum bank was also re-tested, and so we were able to demonstrate the presence, since 2015, of a dual reactivity to Pru p 7 and Pru p 3.

**Conclusion:** Strict avoidance of fruits containing nsLTPs and GRPs fruit before physical exercise. AIT prescribed only for Cypress.

**Case 3 (published²³⁰)**

**Clinical History:** The patient is a 40 years-old man currently living in Paris (northern France) and born in southwest France. He has suffered since childhood from cypress pollen allergy and also food allergy and he experienced an anaphylactic shock after ingestion of pomegranate (*Punica granatum*, Lythraceae family) and strong oral syndrome after ingestion of *Rosaceae* fruits (apple and peach). He has seasonal rhino-conjunctivitis during the cypress and birch pollen seasons, which overlap in the north of France, relieved by antihistaminic treatment.

**Allergy testing:** SPT are positive for birch and cypress pollen extracts. Specific IgE antibodies to birch (27.2 kU/L) and cypress (1.42 kU/L) pollen, citrus (1.38 kU/L), apple (2.62 kU/L), peach (1.78 kU/L), strawberry (0.49 kU/L), kiwi (0.43 kU/L) and cherry (1.99 kU/L) extracts were found with singleplex technology (ImmunoCAP Specific IgE test) and also multiplex microchips (ImmunoCAP ISAC). This patient was studied by immunoblot
<figure><img src="image_316.png" /><figcaption>FIGURE 182 Diagnostic algorithm to assess GRP sensitization</figcaption></figure>

it is possible to in vitro test Pru p 7, and this analysis should always be included in allergy work-out in patients with severe reactions to *Rosaceae*, pomegranate, or citrus fruits.

**Case 2 (original):**

**Clinical History:** Female, Italy, born 1990. The patient has been suffering from seasonal allergic rhinitis between February and March since 2000. After ingestion of a peeled peach and about 30 min running, she had an anaphylactic reaction (low blood pressure, abdominal pain, generalized flushing and swelling, followed by respiratory difficulty due to laryngeal obstruction) and subsequent emergency treatment. Another similar adverse reaction occurred after ingestion of two walnuts associated with moderate physical exercise.

**Allergy testing:** (A) SPT: Environmental allergens: Cypress pollen (*Juniperus a.):* 10mm × 6mm; plane tree (*Platanus a.):* 5mm × 6mm; olive tree (*Olea e.):* 3mm × 2mm; mugwort (*Artemisia v.):* 7mm × 4mm. Food allergens: all negative except walnut (*Juglans r. nut*): 7mm × 5mm and peach (*Prunus p.):* 19mm × 8mm. (B) *In-vitro* testing: [2015] Total IgE 350.3 kU/L, specific IgE to Cypress pollen (*Cupressus a.):* 12.5 kU/L; plane tree (*Platanus a.): 0.66 kU/L; Olive tree (*Olea e.):* 0.12 kU/L; Mugwort (*Artemisia v.):* 2.2 kU/L; Walnut (*Juglans r. nut*) 3.82 kU/L; rPru p 3: 1.79 kU/L.

After 6 years, the patient returned to visit reporting a further reaction after physical exertion (bicycle) performed after ingesting an orange. The patient was studied with a multiplex method, which allowed to highlight, in addition to the already known

reactivity to Cypress (Cry j 1: 2.31 kU/L and Cup a 1: 31.93 kU/L) and nsLTP (Ole e 7: 1.26 kU/L; Cor a 8: 0.94 kU/L; Jug r 3: 0.35 kU/L; Art v 3: 0.52 kU/L; Pru p 3: 2.42 kU/L), also the presence of reactivity to Pru p 7 (8.34 kU/L). Interestingly, the 2015 serum stored in our serum bank was also re-tested, and so we were able to demonstrate the presence, since 2015, of a dual reactivity to Pru p 7 and Pru p 3.

**Conclusion:** Strict avoidance of fruits containing nsLTPs and GRPs fruit before physical exercise. AIT prescribed only for Cypress.

**Case 3 (published<sup>230</sup>)**

**Clinical History:** The patient is a 40 years-old man currently living in Paris (northern France) and born in southwest France. He has suffered since childhood from cypress pollen allergy and also food allergy and he experienced an anaphylactic shock after ingestion of pomegranate (*Punica granatum*, *Lythraceae* family) and strong oral syndrome after ingestion of *Rosaceae* fruits (apple and peach). He has seasonal rhino-conjunctivitis during the cypress and birch pollen seasons, which overlap in the north of France, relieved by antihistaminic treatment.

**Allergy testing:** SPT are positive for birch and cypress pollen extracts. Specific IgE antibodies to birch (27.2 kU/L) and cypress (1.42 kU/L) pollen, citrus (1.38 kU/L), apple (2.62 kU/L), peach (1.78 kU/L), strawberry (0.49 kU/L), kiwi (0.43 kU/L) and cherry (1.99 kU/L) extracts were found with singleplex technology (ImmunoCAP Specific IgE test) and also multiplex microchips (ImmunoCAP ISAC). This patient was studied by immunoblot
against cypress pollen, peach, citrus and pomegranate extracts and Pru p 7 and snakin-1, the GRPs of peach and potato, respectively. All immunoblots were positive at low MW corresponding to a GRP-specific IgE reactivity. Moreover, basophil activation test with total allergen source extracts (cypress pollen, peach and pomegranate) and purified allergens (Cup s 7 and Pru p 7) was found positive in contrast to snakin-1 in keeping with the tolerance to potatoes mentioned by the patient.

**Conclusion:** Strict avoidance of *Rosaceae* fruits and especially pomegranate.

**Case 4** (partially published¹⁵⁷⁷)

**Clinical History:** The patient is a 16-year-old Japanese girl allergic to Japanese cedar (*Cryptomeria japonica*) pollen who suffered from an anaphylactic reaction after consuming chili pepper. She was diagnosed allergic to Japanese cedar pollen when she was 10 years old. She suffered also from an allergy to apple, peach, and orange with symptoms of anaphylaxis exacerbated by physical exercise (or before menstruation) with an onset at the age of 12 years after the consumption of canned peach. At 14 years, consuming a Korean cuisine dish containing beef, bean sprout, spinach, fiddlehead fern, chili pepper, and rice, she developed anaphylaxis with symptoms of facial angioedema, systemic erythema, cough, dyspnea, and cramp. At 16 years, she again experienced a similar reaction after consuming a Chinese cuisine dish containing tofu, minced meat, and chili pepper.

**Allergy testing:** Specific IgE evaluation showed a high titer to Japanese cedar (220 kU/L) and cypress (31.1 kU/L) pollen extracts

together with other pollen and food sensitizations to peach (4.7 kU/L), apple (2.54 kU/L), orange (4.55 kU/L), potato (1.08 kU/L) and confirmed the sensitization to chili pepper (0.24 kU/L). She has no IgE against nsLTPs and a low titer to PR-10.

Oral food challenges to chili pepper (125 mg) or peach (30 g of canned peach) were positive inducing allergic symptoms that include anaphylaxis and required adrenaline and fluid supplement. In agreement, peach and chili pepper extracts were able to ex vivo activate the patient's basophils.

Studied by direct and competitive immunoblot on Japanese cedar pollen proteins, the patient showed IgE reactivities at low MW inhibited not only by Cry j 7, the GRP of Japanese cedar pollen but also by Cap a 7, the GRP from bell pepper.

When tested on bell pepper extracts this patient showed IgE reactivity to a unique cationic LMW *Capsicum annuum* protein from bell and chili pepper pulp extract. The reactivity could be inhibited by Cry j 7, Cap a 7 or Pru p 7. This patient is also allergic to potato, a species from the same family as bell pepper, Solanaceae, and an IgE reactivity was found against recombinant snakin-1, the GRP from potato.

**Conclusion:** This young patient is shown to be sensitized to another member of the GRP family, an allergen as yet undescribed in *Capsicum annuum*, Cap a 7. Allergy to bell/chili pepper is very rare and IgE reactivity to GRP is exceptionally reflecting a very peculiar mechanism of crossed and reinforced specific sensitization. It seems that this allergy is associated with another very rare allergy to the GRP of potato, snakin-1.
against cypress pollen, peach, citrus and pomegranate extracts and Pru p 7 and snakin-1, the GRPs of peach and potato, respectively. All immunoblots were positive at low MW corresponding to a GRP-specific IgE reactivity. Moreover, basophil activation test with total allergen source extracts (cypress pollen, peach and pomegranate) and purified allergens (Cup s 7 and Pru p 7) was found positive in contrast to snakin-1 in keeping with the tolerance to potatoes mentioned by the patient.

**Conclusion:** Strict avoidance of *Rosaceae* fruits and especially pomegranate.

**Case 4 (partially published¹⁵⁷⁷)**

**Clinical History:** The patient is a 16-year-old Japanese girl allergic to Japanese cedar (*Cryptomeria japonica*) pollen who suffered from an anaphylactic reaction after consuming chili pepper. She was diagnosed allergic to Japanese cedar pollen when she was 10 years old. She suffered also from an allergy to apple, peach, and orange with symptoms of anaphylaxis exacerbated by physical exercise (or before menstruation) with an onset at the age of 12 years after the consumption of canned peach. At 14 years, consuming a Korean cuisine dish containing beef, bean sprout, spinach, fiddlehead fern, chili pepper, and rice, she developed anaphylaxis with symptoms of facial angioedema, systemic erythema, cough, dyspnea, and cramp. At 16 years, she again experienced a similar reaction after consuming a Chinese cuisine dish containing tofu, minced meat, and chili pepper.

**Allergy testing:** Specific IgE evaluation showed a high titer to Japanese cedar (220 kU/L) and cypress (31.1 kU/L) pollen extracts

together with other pollen and food sensitizations to peach (4.7 kU/L), apple (2.54 kU/L), orange (4.55 kU/L), potato (1.08 kU/L) and confirmed the sensitization to chili pepper (0.24 kU/L). She has no IgE against nsLTPs and a low titer to PR-10.

Oral food challenges to chili pepper (125 mg) or peach (30 g of canned peach) were positive inducing allergic symptoms that include anaphylaxis and required adrenaline and fluid supplement. In agreement, peach and chili pepper extracts were able to ex vivo activate the patient's basophils.

Studied by direct and competitive immunoblot on Japanese cedar pollen proteins, the patient showed IgE reactivities at low MW inhibited not only by Cry j 7, the GRP of Japanese cedar pollen but also by Cap a 7, the GRP from bell pepper.

When tested on bell pepper extracts this patient showed IgE reactivity to a unique cationic LMW *Capsicum annuum* protein from bell and chili pepper pulp extract. The reactivity could be inhibited by Cry j 7, Cap a 7 or Pru p 7. This patient is also allergic to potato, a species from the same family as bell pepper, Solanaceae, and an IgE reactivity was found against recombinant snakin-1, the GRP from potato.

**Conclusion:** This young patient is shown to be sensitized to another member of the GRP family, an allergen as yet undescribed in *Capsicum annuum*, Cap a 7. Allergy to bell/chili pepper is very rare and IgE reactivity to GRP is exceptionally reflecting a very peculiar mechanism of crossed and reinforced specific sensitization. It seems that this allergy is associated with another very rare allergy to the GRP of potato, snakin-1.
# C10 - Oleosins

## Uta Jappe, Ronald van Ree

### Highlights

* Oleosins have a unique structure: a central hydrophobic domain flanked on each side by relatively hydrophilic domains. Oleosins are lipophilic.

* Therefore they are underrepresented in aqueous extract-based *in vitro*- and *in vivo*-routine diagnostic tests.

* Oleosins are resistant to heat and enzymatic processing.

* An increase in allergenicity has been observed for peanut and hazelnut oleosins after roasting when compared to raw seeds.

## 1 - The protein

In 1998, Olszewski and co-workers reported an allergenic protein present in peanut oil.<sup>1576</sup> After its purification and identification as peanut oleosin, they could show in 2002 IgE-binding in sera of 3 peanut-allergic patients.<sup>1577,1578</sup> Moreover, they provided the first data for increased allergenicity of oleosins from peanuts that were roasted,<sup>1578</sup> which was later confirmed by Schwager et al., 2017.<sup>1236</sup> Oleosins are lipophilic, which is due to the division of the primary sequence into three defined structural domains: a central hydrophobic domain of 72 amino acids flanked on each side by relatively hydrophilic domains.<sup>1579</sup> This feature is unique. It is the hydrophobic domain that is embedded into the oil body membrane whereas the hydrophilic parts reside on the surface and interact with the head groups of phospholipids (Figure 183).<sup>1579-1581</sup> The N-terminal domains are either amphipathic or hydrophilic, the C-terminal part is amphipathic. Oleosins are heat and digestion resistant (Table 102).

The molecular weight ranges from 14 kDa to 17 kDa. A BLAST search of the identified IgE-binding sequences revealed a sequence similarity between oleosins (Figure 184)<sup>1236</sup>. Since this method is limited to the detection of sequential epitopes, conformational epitopes responsible for allergic cross-reactions between oleosins of different sources are not included.

## 2 - The family

Oleosins are stabilizing proteins of the membrane of seed oil bodies, which are lipid storage organelles (Figure 183). They consist of a lipid core, surrounded by a single layer of phospholipids and embedded proteins, the oleosins,caleosins (~30 kDa)<sup>1581-1585</sup> and steroleosins (~40 kDa). Among these proteins, the oleosins represent 80-90% of total protein.<sup>1586</sup> So far, oil bodies have been detected in diverse plants like peanut, walnut, hazelnut, soybean, sesame, maize, rapeseed, and sunflower.<sup>1582</sup> Ten allergens from 4 different plant sources (peanut, hazelnut, sesame, and buckwheat) are presently documented in the WHO/IUIS allergen nomenclature database<sup>1587</sup> (Table 103).

## 3 - Clinical relevance

After a hazelnut oleosin was obtained by cloning in 2006 and hypothesized to be a new allergen,<sup>1587</sup> two oleosins (Cor a 12 and Cor a 13)

<table><caption>TABLE 102 Characteristics of the prototype protein, Ara h 15</caption><thead><tr><th colspan="2">Characteristics of the prototype protein, Ara h 15</th></tr></thead><tbody><tr><td>Allergen source</td><td>Peanuts</td></tr><tr><td>Protein family</td><td>Oleosins</td></tr><tr><td>UniProtKB accession No</td><td>Q647G3</td></tr><tr><td>Three-dimensional structure available</td><td>No</td></tr><tr><td>Molecular structure</td><td>The primary sequence is divided into three defined structural domains: a central hydrophobic domain flanked on either side by relatively hydrophilic domains</td></tr><tr><td>Theoretical molecular weight</td><td>16875,16 Da</td></tr><tr><td>Length</td><td>166 amino acids</td></tr><tr><td>Ligand binding</td><td>Unknown</td></tr><tr><td>Dimerization</td><td>Yes</td></tr><tr><td>Glycosylation</td><td>No sites predicted</td></tr><tr><td>Disulfide bonds</td><td>No</td></tr><tr><td>Isoelectric point</td><td>8.99</td></tr><tr><td>Synthesis</td><td>Details not available</td></tr></tbody></table>

<figure><img src="image_309.png" /><figcaption>FIGURE 183 Schematic model of an oil body with its components (left) and the determination of the molecular mass of oil body proteins from peanut by SDS-polyacrylamide gel electrophoresis (right), taken from<sup>1580</sup></figcaption></figure>
# C10 - Oleosins

## Uta Jappe, Ronald van Ree

### Highlights

* Oleosins have a unique structure: a central hydrophobic domain flanked on each side by relatively hydrophilic domains. Oleosins are lipophilic.

* Therefore they are underrepresented in aqueous extract-based *in vitro*- and *in vivo*-routine diagnostic tests.

* Oleosins are resistant to heat and enzymatic processing.

* An increase in allergenicity has been observed for peanut and hazelnut oleosins after roasting when compared to raw seeds.

### 1 - The protein

In 1998, Olszewski and co-workers reported an allergenic protein present in peanut oil.<sup>1576</sup> After its purification and identification as peanut oleosin, they could show in 2002 IgE-binding in sera of 3 peanut-allergic patients.<sup>1577,1578</sup> Moreover, they provided the first data for increased allergenicity of oleosins from peanuts that were roasted,<sup>1578</sup> which was later confirmed by Schwager et al., 2017.<sup>1236</sup> Oleosins are lipophilic, which is due to the division of the primary sequence into three defined structural domains: a central hydrophobic domain of 72 amino acids flanked on each side by relatively hydrophilic domains.<sup>1579</sup> This feature is unique. It is the hydrophobic domain that is embedded into the oil body membrane whereas the hydrophilic parts reside on the surface and interact with the head groups of phospholipids (Figure 183).<sup>1579-1581</sup> The N-terminal domains are either amphipathic or hydrophilic, the C-terminal part is amphipathic. Oleosins are heat and digestion resistant (Table 102).

The molecular weight ranges from 14 kDa to 17 kDa. A BLAST search of the identified IgE-binding sequences revealed a sequence similarity between oleosins (Figure 184)<sup>1236</sup>. Since this method is limited to the detection of sequential epitopes, conformational epitopes responsible for allergic cross-reactions between oleosins of different sources are not included.

### 2 - The family

Oleosins are stabilizing proteins of the membrane of seed oil bodies, which are lipid storage organelles (Figure 183). They consist of a lipid core, surrounded by a single layer of phospholipids and embedded proteins, the oleosins,caleosins (~30 kDa)<sup>1581-1585</sup> and steroleosins (~40 kDa). Among these proteins, the oleosins represent 80-90% of total protein.<sup>1586</sup> So far, oil bodies have been detected in diverse plants like peanut, walnut, hazelnut, soybean, sesame, maize, rapeseed, and sunflower.<sup>1582</sup> Ten allergens from 4 different plant sources (peanut, hazelnut, sesame, and buckwheat) are presently documented in the WHO/ IUIS allergen nomenclature database<sup>1587</sup> (Table 103).

### 3 - Clinical relevance

After a hazelnut oleosin was obtained by cloning in 2006 and hypothesized to be a new allergen,<sup>1587</sup> two oleosins (Cor a 12 and Cor a 13)

<table><caption>TABLE 102 Characteristics of the prototype protein, Ara h 15</caption><thead><tr><th colspan="2">Characteristics of the prototype protein, Ara h 15</th></tr></thead><tbody><tr><td>Allergen source</td><td>Peanuts</td></tr><tr><td>Protein family</td><td>Oleosins</td></tr><tr><td>UniProtKB accession No</td><td>Q647G3</td></tr><tr><td>Three-dimensional structure available</td><td>No</td></tr><tr><td>Molecular structure</td><td>The primary sequence is divided into three defined structural domains: a central hydrophobic domain flanked on either side by relatively hydrophilic domains</td></tr><tr><td>Theoretical molecular weight</td><td>16875,16 Da</td></tr><tr><td>Length</td><td>166 amino acids</td></tr><tr><td>Ligand binding</td><td>Unknown</td></tr><tr><td>Dimerization</td><td>Yes</td></tr><tr><td>Glycosylation</td><td>No sites predicted</td></tr><tr><td>Disulfide bonds</td><td>No</td></tr><tr><td>Isoelectric point</td><td>8.99</td></tr><tr><td>Synthesis</td><td>Details not available</td></tr></tbody></table>

<figure><img src="image_317.png" /><figcaption>FIGURE 183 Schematic model of an oil body with its components (left) and the determination of the molecular mass of oil body proteins from peanut by SDS-polyacrylamide gel electrophoresis (right), taken from<sup>1580</sup></figcaption></figure>
<figure><img src="image_310.png" /><figcaption>FIGURE 184 Similarity of sequences between oleosins in % (all isoforms included) that are documented in www.allergen.org. Melanie Plum, PhD, Research Group Uta Jappe, Research Center Borstel, Germany.</figcaption></figure>

<table><caption>TABLE 103 Oleosins documented by the WHO/IUIS allergen nomenclature subcommittee [¹⁵⁸⁰, modified]</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th><th>Molecular weight (kDa)</th></tr></thead><tbody><tr><td rowspan="4">Fabales</td><td rowspan="4">Peanut (Arachis hypogaea) [1236, 1578, 1590]</td><td>Ara h 10</td><td>16</td></tr><tr><td>Ara h 11</td><td>14</td></tr><tr><td>Ara h 14</td><td>17.5</td></tr><tr><td>Ara h 15</td><td>17</td></tr><tr><td rowspan="3">Fagales</td><td rowspan="3">Hazelnut (Corylus avellana) [62, 1588, 1589]</td><td>Cor a 12</td><td>17</td></tr><tr><td>Cor a 13</td><td>14-16</td></tr><tr><td>Cor a 15</td><td>17</td></tr><tr><td rowspan="2">Lamiales</td><td rowspan="2">Sesame (Sesamum indicum) [1593, 1600]</td><td>Ses i 4</td><td>17</td></tr><tr><td>Ses i 5</td><td>15</td></tr><tr><td>Caryophyllales</td><td>Tartarian buckwheat (Fagopyrum tataricum) [1591, 1592]</td><td>Fag t 6</td><td>18</td></tr></tbody></table>

were subsequently shown to be allergenic in 2014⁶² and accepted by the WHO/IUIS allergen nomenclature subcommittee.¹⁵⁸⁷ In 2021, a third was described, Cor a 15.¹⁵⁸⁹ Cor a 15 was the oleosin that was predominantly bound by IgE in an Italian cohort of hazelnut-allergic children. According to the authors, some allergic children recognized solely oleosins by their IgE, which so far is in contrast to patients with peanut and sesame allergy. In this case, hazelnut oleosins would enrich molecular allergy diagnostics immensely in a subpopulation of hazelnut-allergic children.¹⁵⁸⁶

In 2015, the allergenicity of Ara h 10 and Ara h 11 from peanut was published together with the identification of additional allergenic peanut oleosins, Ara h 14 and Ara h 15. The allergenic potential of the oleosin initially observed in peanut oil¹²³⁶,¹⁵⁷⁶,¹⁵⁹⁰,¹⁶²⁶ was now shown in a larger cohort of peanut-allergic subjects.¹²³⁶ Most probably, oleosins from other plant species (soybean, flax, walnut,

sunflower [summarized in¹⁵⁸⁰]) might cause allergic reactions, as well. An IgE-binding epitope of Ara h 15 was shown to be cross-reactive with buckwheat,¹⁵⁹¹ which suggested oleosins to be present in this source. This was recently confirmed.¹⁵⁹² In addition to the general feature of allergenicity, oleosins from peanut, hazelnut and sesame have been associated with severe allergic reactions.⁶²,¹²³⁶,¹⁵⁹⁰,¹⁵⁹³ This has been confirmed in 2017 for a cohort of peanut-allergic patients (of meanwhile above n=70), where only those with severe reactions to peanut and not those with mild or moderate symptoms, had IgE to oleosins. Although these patients were also IgE-positive for Ara h 2, this points to oleosins as marker allergens for the severity of a reaction,¹²³⁶ and so far, foods known to contain allergens from the oleosin family are requiring labelling on food products. It was shown that roasting increased IgE binding to peanut oleosins¹²³⁶ and hazelnut oleosins.¹⁵⁸⁹
<figure><img src="image_318.png" /><figcaption>FIGURE 184 Similarity of sequences between oleosins in % (all isoforms included) that are documented in www.allergen.org. Melanie Plum, PhD, Research Group Uta Jappe, Research Center Borstel, Germany.</figcaption></figure>

<table><caption>TABLE 103 Oleosins documented by the WHO/IUIS allergen nomenclature subcommittee [¹⁵⁸⁰, modified]</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th><th>Molecular weight (kDa)</th></tr></thead><tbody><tr><td rowspan="4">Fabales</td><td rowspan="4">Peanut (Arachis hypogaea) [1236, 1578, 1590]</td><td>Ara h 10</td><td>16</td></tr><tr><td>Ara h 11</td><td>14</td></tr><tr><td>Ara h 14</td><td>17.5</td></tr><tr><td>Ara h 15</td><td>17</td></tr><tr><td rowspan="3">Fagales</td><td rowspan="3">Hazelnut (Corylus avellana) [62, 1588, 1589]</td><td>Cor a 12</td><td>17</td></tr><tr><td>Cor a 13</td><td>14-16</td></tr><tr><td>Cor a 15</td><td>17</td></tr><tr><td rowspan="2">Lamiales</td><td rowspan="2">Sesame (Sesamum indicum) [1593, 1600]</td><td>Ses i 4</td><td>17</td></tr><tr><td>Ses i 5</td><td>15</td></tr><tr><td>Caryophyllales</td><td>Tartarian buckwheat (Fagopyrum tataricum) [1591, 1592]</td><td>Fag t 6</td><td>18</td></tr></tbody></table>

were subsequently shown to be allergenic in 2014⁶² and accepted by the WHO/IUIS allergen nomenclature subcommittee.¹⁵⁸⁷ In 2021, a third was described, Cor a 15.¹⁵⁸⁹ Cor a 15 was the oleosin that was predominantly bound by IgE in an Italian cohort of hazelnut-allergic children. According to the authors, some allergic children recognized solely oleosins by their IgE, which so far is in contrast to patients with peanut and sesame allergy. In this case, hazelnut oleosins would enrich molecular allergy diagnostics immensely in a subpopulation of hazelnut-allergic children.¹⁵⁸⁶

In 2015, the allergenicity of Ara h 10 and Ara h 11 from peanut was published together with the identification of additional allergenic peanut oleosins, Ara h 14 and Ara h 15. The allergenic potential of the oleosin initially observed in peanut oil¹²³⁶,¹⁵⁷⁶,¹⁵⁹⁰,¹⁶²⁶ was now shown in a larger cohort of peanut-allergic subjects.¹²³⁶ Most probably, oleosins from other plant species (soybean, flax, walnut,

sunflower [summarized in¹⁵⁸⁰]) might cause allergic reactions, as well. An IgE-binding epitope of Ara h 15 was shown to be cross-reactive with buckwheat,¹⁵⁹¹ which suggested oleosins to be present in this source. This was recently confirmed.¹⁵⁹² In addition to the general feature of allergenicity, oleosins from peanut, hazelnut and sesame have been associated with severe allergic reactions.⁶²,¹²³⁶,¹⁵⁹⁰,¹⁵⁹³ This has been confirmed in 2017 for a cohort of peanut-allergic patients (of meanwhile above n=70), where only those with severe reactions to peanut and not those with mild or moderate symptoms, had IgE to oleosins. Although these patients were also IgE-positive for Ara h 2, this points to oleosins as marker allergens for the severity of a reaction,¹²³⁶ and so far, foods known to contain allergens from the oleosin family are requiring labelling on food products. It was shown that roasting increased IgE binding to peanut oleosins¹²³⁶ and hazelnut oleosins.¹⁵⁸⁹
Ehlers and co-workers investigated the diagnostic relevance of recombinantly expressed and native sesame oleosins. They observed no allergic individuals with an oleosin mono-sensitization but always a simultaneous recognition of other sesame allergens.¹⁵⁹⁵ In addition, nonallergic individuals also had IgE to sesame oleosins, which is different for peanut oleosins that were only recognized by IgE in sera from patients with severe peanut allergy.¹²³⁶ Up to 30% of sesame allergic patients cannot be diagnosed by routine allergy diagnostic tests, and according to Ehlers and co-workers, they will at present not profit from the addition of Ses i 4 and Ses i 5 to the diagnostic allergen panel.¹⁵⁹⁵ However, it is plausible that the application step to eliminate impurities of seed storage proteins from the oleosin fraction has maybe led to the disruption of lipids, which may be important for the recognition of sesame oleosins.

Cross-reactivity was hypothesized from the observation that an IgE-binding amino acid sequence in the C-terminal part of the Ara h 15 is also found with a high degree of homology in oleosins from several other food allergen sources, such as hazelnut, rapeseed, soy, sesame, and almond.¹²³⁶

In the large EuroPrevall outpatient clinic survey in 12 European cities, 13% of patients reporting hazelnut allergy were sensitized to nCor a 12. Although the prevalence of sensitization was higher in patients reporting severe symptoms than in those with mild to moderate symptoms, a significant association with severity was not demonstrated.⁴⁵

## 4 – Clinical management

**Clinical diagnosis:** Patients with severe food allergic reactions to nuts, legumes and seeds are presently investigated for IgE against storage proteins and/or lipid transfer proteins because these allergens are widely accepted as potential markers. However, concerning anaphylactic reactions to lipid-rich representatives of these food allergen sources, there has been an increasing suspicion that severe reactions may also be associated with oleosins. Support for this has recently been reported.¹²³⁶ Unfortunately, the lipophilic nature of this family of proteins has hampered the development of reliable CRD reagents for oleosins, both singleplex and array-based until recently.¹²³⁶,¹⁵⁹⁶ There are now reports on recombinant oleosins being used in arrays, as they are soluble in aqueous solutions.

**Case history:** In general, it can be recommended to test IgE against oleosins if available.

**Skin prick test:** Oleosins will be underrepresented in skin prick test solutions⁶² since these are also based on aqueous solutions.

Only a prick-to-prick test with native foods can be expected to include natural oleosins. In this regard, “Tahini” sauce may be helpful as it allows *in vivo* detection of sesame sensitization. It may even be helpful for the diagnosis of peanut allergic individuals without IgE against Ara h 2 and sesame allergic individuals without IgE to seed storage proteins.¹⁵⁹⁷

**IgE-detection assay:** So far, a recombinant oleosin from peanut is now applied on the ALEX2 array⁴⁴,¹⁵⁹⁸ (A naturally purified hazelnut oleosin was used in ImmunoCAP in the investigation by Datema et al.⁴⁴ but is not yet commercially available).

**Treatment:** The treatment of severe reactions to foods containing oleosins is still strict avoidance. The patients should be provided with an emergency kit. So far, only for peanut allergy, oral allergen immunotherapy has recently been authorized (see chapter B18).

## 5 – Clinical cases

**Clinical history:** An eight-month-old girl with generalized atopic eczema (SCORAD 67) who had been solely breastfed experienced sin-cere flare-ups of eczema a couple of days after coming home from the Dermatology clinic where she had been treated. Eventually, a trigger of eczema was suspected in the domestic situation. A thorough environmental history revealed that peanut butter was regularly consumed, a habit introduced by the mother who was US American. The child itself had never consumed peanuts in any form.¹⁵⁹⁹

**Diagnostic and therapeutic considerations:** The allergens contained in peanut butter were suspected to be responsible for the worsening of her eczema, whenever she returned home from the hospital. An atopy patch test was performed and confirmed peanut to induce eczema. The investigation for a filaggrin mutation was negative. First series of IgE tests—Total serum IgE>100IU/L; IgE to peanut extract: 71 kU/L. Further IgE tests—Molecular allergy diagnostics revealed IgE to Ara h 1: >100 kU/L; Ara h 2: 41 kU/L; Ara h 3: 24.7 kU/L; Ara h 6: >100 kU/L; Ara h 8: 0.1 kU/L; Ara h 9: 0.49 kU/L.

Testing IgE to new allergen molecules—Several years later, after peanut oleosins had been identified and purified by us, her serum sampled for the first investigations was tested positive in immunoblot for IgE against the peanut oleosins Ara h 10, Ara h 11 and Ara h 14, Ara h 15.¹²³⁶

**Diagnosis and therapy:** The fact that without consumption of peanuts such a strong sensitization towards storage proteins, defensins and oleosins, all associated with severe allergic reactions to peanut consumption had taken place strongly speaks in favor of a cutaneous sensitization. Peanut butter was removed from the household, the house was thoroughly and repeatedly cleaned.
Ehlers and co-workers investigated the diagnostic relevance of recombinantly expressed and native sesame oleosins. They observed no allergic individuals with an oleosin mono-sensitization but always a simultaneous recognition of other sesame allergens.<sup>1595</sup> In addition, nonallergic individuals also had IgE to sesame oleosins, which is different for peanut oleosins that were only recognized by IgE in sera from patients with severe peanut allergy.<sup>1236</sup> Up to 30% of sesame allergic patients cannot be diagnosed by routine allergy diagnostic tests, and according to Ehlers and co-workers, they will at present not profit from the addition of Ses i 4 and Ses i 5 to the diagnostic allergen panel.<sup>1595</sup> However, it is plausible that the application step to eliminate impurities of seed storage proteins from the oleosin fraction has maybe led to the disruption of lipids, which may be important for the recognition of sesame oleosins.

Cross-reactivity was hypothesized from the observation that an IgE-binding amino acid sequence in the C-terminal part of the Ara h 15 is also found with a high degree of homology in oleosins from several other food allergen sources, such as hazelnut, rapeseed, soy, sesame, and almond.<sup>1236</sup>

In the large EuroPrevall outpatient clinic survey in 12 European cities, 13% of patients reporting hazelnut allergy were sensitized to nCor a 12. Although the prevalence of sensitization was higher in patients reporting severe symptoms than in those with mild to moderate symptoms, a significant association with severity was not demonstrated.<sup>45</sup>

## 4 – Clinical management

**Clinical diagnosis:** Patients with severe food allergic reactions to nuts, legumes and seeds are presently investigated for IgE against storage proteins and/or lipid transfer proteins because these allergens are widely accepted as potential markers. However, concerning anaphylactic reactions to lipid-rich representatives of these food allergen sources, there has been an increasing suspicion that severe reactions may also be associated with oleosins. Support for this has recently been reported.<sup>1236</sup> Unfortunately, the lipophilic nature of this family of proteins has hampered the development of reliable CRD reagents for oleosins, both singleplex and array-based until recently.<sup>1236,1596</sup> There are now reports on recombinant oleosins being used in arrays, as they are soluble in aqueous solutions.

**Case history:** In general, it can be recommended to test IgE against oleosins if available.

**Skin prick test:** Oleosins will be underrepresented in skin prick test solutions<sup>62</sup> since these are also based on aqueous solutions.

Only a prick-to-prick test with native foods can be expected to include natural oleosins. In this regard, “Tahini” sauce may be helpful as it allows *in vivo* detection of sesame sensitization. It may even be helpful for the diagnosis of peanut allergic individuals without IgE against Ara h 2 and sesame allergic individuals without IgE to seed storage proteins.<sup>1597</sup>

**IgE-detection assay:** So far, a recombinant oleosin from peanut is now applied on the ALEX2 array<sup>44,1598</sup> (A naturally purified hazelnut oleosin was used in ImmunoCAP in the investigation by Datema et al.<sup>44</sup> but is not yet commercially available).

**Treatment:** The treatment of severe reactions to foods containing oleosins is still strict avoidance. The patients should be provided with an emergency kit. So far, only for peanut allergy, oral allergen immunotherapy has recently been authorized (see chapter B18).

## 5 – Clinical cases

**Clinical history:** An eight-month-old girl with generalized atopic eczema (SCORAD 67) who had been solely breastfed experienced sin-cere flare-ups of eczema a couple of days after coming home from the Dermatology clinic where she had been treated. Eventually, a trigger of eczema was suspected in the domestic situation. A thorough environmental history revealed that peanut butter was regularly consumed, a habit introduced by the mother who was US American. The child itself had never consumed peanuts in any form.<sup>1599</sup>

**Diagnostic and therapeutic considerations:** The allergens contained in peanut butter were suspected to be responsible for the worsening of her eczema, whenever she returned home from the hospital. An atopy patch test was performed and confirmed peanut to induce eczema. The investigation for a filaggrin mutation was negative. First series of IgE tests—Total serum IgE>100IU/L; IgE to peanut extract: 71 kU/L. Further IgE tests—Molecular allergy diagnostics revealed IgE to Ara h 1: >100 kU/L; Ara h 2: 41 kU/L; Ara h 3: 24.7 kU/L; Ara h 6: >100 kU/L; Ara h 8: 0.1 kU/L; Ara h 9: 0.49 kU/L.

Testing IgE to new allergen molecules—Several years later, after peanut oleosins had been identified and purified by us, her serum sampled for the first investigations was tested positive in immunoblot for IgE against the peanut oleosins Ara h 10, Ara h 11 and Ara h 14, Ara h 15.<sup>1236</sup>

**Diagnosis and therapy:** The fact that without consumption of peanuts such a strong sensitization towards storage proteins, defensins and oleosins, all associated with severe allergic reactions to peanut consumption had taken place strongly speaks in favor of a cutaneous sensitization. Peanut butter was removed from the household, the house was thoroughly and repeatedly cleaned.
<figure><img src="image_311.png" /><figcaption>FIGURE 185 Decision algorithm</figcaption></figure>

Absolute avoidance of peanuts in the future was advised for the little girl (Figure 185).

**Most important protein or family characteristics**

Oleosins are lipophilic and therefore not present in aqueous extract-based routine diagnostic *in vitro* and *in vivo* tests.⁶² They are resistant to heat and enzymatic processing. An increase in allergenicity has been observed for peanut and hazelnut oleosins after roasting when compared to raw seeds.¹²³⁶,¹⁵⁸⁹

**Remarkable or important clinical aspects**

* Oleosins are potential marker allergens for allergy severity after peanut and hazelnut consumption.⁶²,¹²³⁶,¹⁵⁸⁹

* Risk assessment of anaphylaxis is possible by the detection of IgE to oleosins.

* Precision medicine optimization may make risky and expensive oral challenge tests superfluous.

* The basophil activation test with peanut oleosins truly discriminates between allergic and sensitized individuals.¹²³⁶,¹⁵⁹⁶
<figure><img src="image_319.png" /><figcaption>FIGURE 185 Decision algorithm</figcaption></figure>

Absolute avoidance of peanuts in the future was advised for the little girl (Figure 185).

**Most important protein or family characteristics**

Oleosins are lipophilic and therefore not present in aqueous extract-based routine diagnostic *in vitro* and *in vivo* tests.⁶² They are resistant to heat and enzymatic processing. An increase in allergenicity has been observed for peanut and hazelnut oleosins after roasting when compared to raw seeds.¹²³⁶,¹⁵⁸⁹

**Remarkable or important clinical aspects**

* Oleosins are potential marker allergens for allergy severity after peanut and hazelnut consumption.⁶²,¹²³⁶,¹⁵⁸⁹

* Risk assessment of anaphylaxis is possible by the detection of IgE to oleosins.

* Precision medicine optimization may make risky and expensive oral challenge tests superfluous.

* The basophil activation test with peanut oleosins truly discriminates between allergic and sensitized individuals.¹²³⁶,¹⁵⁹⁶
# C11 - Parvalbumins

Denise Schrama, Tanja Kalic, Martine Morisset, Lars K. Poulsen, Annette Kuehn

## Highlights

* Food, respiratory and contact allergens.

* Fish pan-allergens resistant to food processing.

* Clinical cross-reactivity is based on the presence of highly conserved IgE epitopes.

* Low cross-reactivity between beta-parvalbumins from bony fish and alpha-parvalbumins from cartilaginous fish.

* Monosensitized patients have IgE to species-specific epitopes.

## 1 - The protein

In the early seventies, Gad c 1 was the first parvalbumin identified as major fish allergen in Baltic cod (Gadus callarius).<sup>1609</sup> Subsequent cloning and biomolecular studies were performed with the parvalbumin Gad m 1, the homologous allergen from Atlantic cod (Gadus morhua) (Table 104).<sup>195,1015</sup> Gad m 1 is used as a representative allergenic parvalbumin in many studies. Currently, two isoallergens are listed in the official allergen nomenclature database (www.allergen.org), Gad m 1.01 and Gad m 1.02. Each isoallergen has been characterized as two isoforms (variants) of high sequence identity. The protein structure of cod parvalbumin was first modeled on the basis of the x-ray structure from carp parvalbumin, Cyp c 1. In 2014, the NMR-based protein structure was published, revealing the important characteristics on the folding and stability of parvalbumins.<sup>1610</sup> Gad m 1 has a six alpha-helical protein fold, which is a common feature of parvalbumins (Figure 186). Since then, structures of several other allergenic fish and non-fish parvalbumins were determined.

Gad m 1 was identified as the major allergen in cod muscle. However, food allergy to codfish can also be caused by other allergens, such as Gad m 2 (cod enolase) and Gad m 3 (cod aldolase) (see Chapter B12).<sup>1024</sup> Cod parvalbumin, a highly heat-stable protein of low molecular weight (10–12 kDa), binds Ca2+-ions (or Mg2+-ions) via two loops called EF-hand motifs.<sup>1611</sup> It is involved in the regulation of the intracellular calcium concentration during muscle relaxation.<sup>1611</sup> As all bony fish, cod has two types of muscles, light and dark, which differ by their physiological function. Cod belongs to the whitefish and has mainly light muscle tissue and only a small strip of dark tissue underneath the skin. Parvalbumins are more abundant in the light muscles than the dark ones as described for tuna.<sup>1612</sup> In cod, the parvalbumin level is up to 2 mg per g of muscle tissue (see chapter B12 “Fish allergy” for information on other species).<sup>1009</sup> B cell epitopes have been determined for cod parvalbumin.<sup>1015,1613</sup> Several regions of the protein seem to be involved in the antigen-antibody interaction. A correlation was found between the severity of the allergic reaction and the number of epitopes recognized by patients' IgE. Allergic patients who recognized ten IgE-binding

<table><caption>TABLE 104 Basic protein characteristics of Gad m 1</caption><thead><tr><th colspan="2">Basic protein characteristics of Gad m 1</th></tr></thead><tbody><tr><td>Allergen source</td><td>Gadus morhua, Atlantic cod</td></tr><tr><td>Protein family</td><td>Parvalbumin</td></tr><tr><td>UniProtKB accession No</td><td>Q90YL0</td></tr><tr><td>Three-dimensional structure available</td><td>Yes</td></tr><tr><td>Molecular structure</td><td>Alpha-helical structure</td></tr><tr><td>Theoretical molecular weight</td><td>11.55 kDa</td></tr><tr><td>Molecular weight measured by mass spectrometry</td><td>11.36 kDa</td></tr><tr><td>Length</td><td>109 amino acids residues</td></tr><tr><td>Ligand binding</td><td>Yes (Ca2+, Mg2+)</td></tr><tr><td>Oligomerization</td><td>Dimers, oligomers</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>No</td></tr><tr><td>Isoelectric point</td><td>4.58</td></tr><tr><td>Synthesis</td><td>Muscle tissue, cytosolic protein</td></tr><tr><td>Distribution</td><td>Muscle, swimbladder</td></tr></tbody></table>

<figure><img src="image_312.png" /><figcaption>FIGURE 186 A ribbon model of the three-dimensional structure of cod parvalbumin Gad m 1 (PDB: 2MBX). Two calcium ions (purple) are bound by two functional EF-hand motifs. The model was created by ChimeraX (http://www.cgl.ucsf.edu/chime/rax/).</figcaption></figure>

peptides including an important C-terminal epitope had more severe reactions than others.<sup>1613</sup> It was concluded that the number of linear epitopes could serve as a marker for the severity of the allergic reaction. A strategy for immunotherapy using hypoallergenic parvalbumin has been developed but is unavailable for clinical practice.<sup>429,1614</sup>
# C11 - Parvalbumins

Denise Schrama, Tanja Kalic, Martine Morisset, Lars K. Poulsen, Annette Kuehn

## Highlights

* Food, respiratory and contact allergens.

* Fish pan-allergens resistant to food processing.

* Clinical cross-reactivity is based on the presence of highly conserved IgE epitopes.

* Low cross-reactivity between beta-parvalbumins from bony fish and alpha-parvalbumins from cartilaginous fish.

* Monosensitized patients have IgE to species-specific epitopes.

## 1 - The protein

In the early seventies, Gad c 1 was the first parvalbumin identified as major fish allergen in Baltic cod (Gadus callarius).<sup>1609</sup> Subsequent cloning and biomolecular studies were performed with the parvalbumin Gad m 1, the homologous allergen from Atlantic cod (Gadus morhua) (Table 104).<sup>195,1015</sup> Gad m 1 is used as a representative allergenic parvalbumin in many studies. Currently, two isoallergens are listed in the official allergen nomenclature database (www.allergen.org), Gad m 1.01 and Gad m 1.02. Each isoallergen has been characterized as two isoforms (variants) of high sequence identity. The protein structure of cod parvalbumin was first modeled on the basis of the x-ray structure from carp parvalbumin, Cyp c 1. In 2014, the NMR-based protein structure was published, revealing the important characteristics on the folding and stability of parvalbumins.<sup>1610</sup> Gad m 1 has a six alpha-helical protein fold, which is a common feature of parvalbumins (Figure 186). Since then, structures of several other allergenic fish and non-fish parvalbumins were determined.

Gad m 1 was identified as the major allergen in cod muscle. However, food allergy to codfish can also be caused by other allergens, such as Gad m 2 (cod enolase) and Gad m 3 (cod aldolase) (see Chapter B12).<sup>1024</sup> Cod parvalbumin, a highly heat-stable protein of low molecular weight (10–12 kDa), binds Ca2+-ions (or Mg2+-ions) via two loops called EF-hand motifs.<sup>1611</sup> It is involved in the regulation of the intracellular calcium concentration during muscle relaxation.<sup>1611</sup> As all bony fish, cod has two types of muscles, light and dark, which differ by their physiological function. Cod belongs to the whitefish and has mainly light muscle tissue and only a small strip of dark tissue underneath the skin. Parvalbumins are more abundant in the light muscles than the dark ones as described for tuna.<sup>1612</sup> In cod, the parvalbumin level is up to 2 mg per g of muscle tissue (see chapter B12 “Fish allergy” for information on other species).<sup>1009</sup> B cell epitopes have been determined for cod parvalbumin.<sup>1015,1613</sup> Several regions of the protein seem to be involved in the antigen-antibody interaction. A correlation was found between the severity of the allergic reaction and the number of epitopes recognized by patients' IgE. Allergic patients who recognized ten IgE-binding

<table><caption>TABLE 104 Basic protein characteristics of Gad m 1</caption><thead><tr><th colspan="2">Basic protein characteristics of Gad m 1</th></tr></thead><tbody><tr><td>Allergen source</td><td>Gadus morhua, Atlantic cod</td></tr><tr><td>Protein family</td><td>Parvalbumin</td></tr><tr><td>UniProtKB accession No</td><td>Q90YL0</td></tr><tr><td>Three-dimensional structure available</td><td>Yes</td></tr><tr><td>Molecular structure</td><td>Alpha-helical structure</td></tr><tr><td>Theoretical molecular weight</td><td>11.55 kDa</td></tr><tr><td>Molecular weight measured by mass spectrometry</td><td>11.36 kDa</td></tr><tr><td>Length</td><td>109 amino acids residues</td></tr><tr><td>Ligand binding</td><td>Yes (Ca2+, Mg2+)</td></tr><tr><td>Oligomerization</td><td>Dimers, oligomers</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>No</td></tr><tr><td>Isoelectric point</td><td>4.58</td></tr><tr><td>Synthesis</td><td>Muscle tissue, cytosolic protein</td></tr><tr><td>Distribution</td><td>Muscle, swimbladder</td></tr></tbody></table>

<figure><img src="image_320.png" /><figcaption>FIGURE 186 A ribbon model of the three-dimensional structure of cod parvalbumin Gad m 1 (PDB: 2MBX). Two calcium ions (purple) are bound by two functional EF-hand motifs. The model was created by ChimeraX (http://www.cgl.ucsf.edu/chime/rax/).</figcaption></figure>

peptides including an important C-terminal epitope had more severe reactions than others.<sup>1613</sup> It was concluded that the number of linear epitopes could serve as a marker for the severity of the allergic reaction. A strategy for immunotherapy using hypoallergenic parvalbumin has been developed but is unavailable for clinical practice.<sup>429,1614</sup>
So far, successful oral immunotherapy for allergy to cod has only been reported for patients treated with boiled cod.¹⁶¹⁵

## 2 – The family

Based on their protein characteristics, parvalbumins are attributed to two different phylogenetic origins, the alpha- and the beta- lineage.¹⁰³¹ Both subtypes can be found in different organs (central nervous system, endocrine tissue), but the highest expression rates have been determined in muscles.³²⁹ Muscles from mammals and birds express alpha-parvalbumins, which are considered as rarely al- lergenic proteins.¹⁹⁵

Parvalbumins of the beta-subtype have been characterized as pan-allergens in fish muscle.¹⁰³¹ They belong to the EF-hand protein superfamily comprising important allergens from both animal and plant origin (see also chapter C06 “Polcalcins”). These proteins share conserved domains consisting of Ca2+-binding peptide loops flanked on both sides by α-helices. These structures are called EF- hand motifs as both α-helices are arranged like the thumb and the

forefinger of a hand. Fish parvalbumins have three EF-hand motifs (AB, CD, EF) but only the CD- and EF-motifs are functional and bind divalent cations (Ca2+, Mg2+). Parvalbumin structures rearrange globally upon ion-binding or release. The ion-binding capacity is essential for the correct parvalbumin function and its IgE-binding capability.⁴³⁰ Parvalbumins with unfunctional calcium-binding sites bind IgE antibodies from fish-allergic patients only weakly.³²⁹ Therefore, the functional calcium-binding motifs (CD, EF) have been suggested as important conformational B cell epitopes.

### Textbox Parvalbumin family characteristics

* Parvalbumins are members of the Ca2+-binding EF- hand superfamily.

* Clinical cross-reactivity is based on the presence of highly conserved IgE epitopes.

* Monosensitized patients have IgE to species-specific epitopes.

<table><caption>TABLE 105 Gad m 1 - homologus allergens from several fish and non-fish species</caption><thead><tr><td>Taxonomic order</td><td>Allergen source</td><td>Allergen</td></tr></thead><tbody><tr><td>Anura</td><td>Edible frog (Rana esculenta/Pelophylax esculentus)</td><td>Ran e 2*</td></tr><tr><td>Carcharhiniformes</td><td>Edible shark (Mustelus griseus)</td><td>Mus g 1</td></tr><tr><td rowspan="2">Clupeiformes</td><td>Atlantic herring (Clupea harengus)</td><td>Clu h 1*</td></tr><tr><td>Pacific pilchard (Sardinops sagax)</td><td>Sar sa 1*</td></tr><tr><td>Crocodylia</td><td>Australian saltwater crocodile (Crocodylus porosus)</td><td>Cro p 1*</td></tr><tr><td rowspan="2">Cypriniformes</td><td>Grass carp (Ctenopharyngodon idella)</td><td>Cten i 1*</td></tr><tr><td>Common carp (Cyprinus carpio)</td><td>Cyp c 1*</td></tr><tr><td rowspan="5">Gadiformes</td><td>Baltic cod ( Gadus callarias )</td><td>Gad c 1*</td></tr><tr><td>Atlantic cod ( Gadus morhua )</td><td>Gad m 1*</td></tr><tr><td>Atlantic hake ( Merluccius merluccius )</td><td>Mer mr 1</td></tr><tr><td>Alaska pollock ( Gadus chalcogrammus )</td><td>The c 1</td></tr><tr><td>Alaska pollock (Thunnus albacares)</td><td>Gal d 8*</td></tr><tr><td>Galliformes</td><td>Chicken (Gallus gallus domesticus)</td><td>Gal d 8*</td></tr><tr><td rowspan="3">Perciformes</td><td>Barramundi (Lates calcarifer)</td><td>Lat c 1*</td></tr><tr><td>Yellowfin tuna (Thunnus albacares)</td><td>Thu a 1*</td></tr><tr><td>Swordfish (Xiphias gladius)</td><td>Xip g 1*</td></tr><tr><td rowspan="2">Pleuronectiformes</td><td>Megrim (Lepidorhombus whiffiagonis)</td><td>Lep w 1*</td></tr><tr><td>Common sole (Solea solea)</td><td>Sole s 1*</td></tr><tr><td rowspan="2">Salmoniformes</td><td>Rainbow trout (Onchorhynchus mykiss)</td><td>Onc m 1*</td></tr><tr><td>Atlantic salmon (Salmo salar)</td><td>Sal s 1*</td></tr><tr><td rowspan="2">Scombriformes</td><td>Indian mackerel (Rastrelliger kanagurta)</td><td>Ras k 1*</td></tr><tr><td>Atlantic mackerel (Scomber scrombus)</td><td>Sco s 1*</td></tr><tr><td>Scorpaeniformes</td><td>Redfish (Sebastes marinus)</td><td>Seb m 1*</td></tr><tr><td>Siluriformes</td><td>Striped catfish (Pangasianodon hypophtalmus)</td><td>Pan h 1*</td></tr></tbody></table>

<footnote>* Allergens officially accepted and designated by the WHO/IUIS Allergen Nomenclature Sub-Committee (www.allergen.org); all others are unofficial names taken from the Allergome database (www.allergome.org).</footnote>

<footnote>* Allergens officially accepted and designated by the WHO/IUIS Allergen Nomenclature Sub-Committee (http://www.allergen.org); all others are unofficial names taken from the Allergome database (http://www.allergome.org).</footnote>
So far, successful oral immunotherapy for allergy to cod has only been reported for patients treated with boiled cod.<sup>1615</sup>

## 2 – The family

Based on their protein characteristics, parvalbumins are attributed to two different phylogenetic origins, the alpha- and the beta- lineage.<sup>1031</sup> Both subtypes can be found in different organs (central nervous system, endocrine tissue), but the highest expression rates have been determined in muscles.<sup>329</sup> Muscles from mammals and birds express alpha-parvalbumins, which are considered as rarely al- lergenic proteins.<sup>195</sup>

Parvalbumins of the beta-subtype have been characterized as pan-allergens in fish muscle.<sup>1031</sup> They belong to the EF-hand protein superfamily comprising important allergens from both animal and plant origin (see also chapter C06 “Polcalcins”). These proteins share conserved domains consisting of Ca2+-binding peptide loops flanked on both sides by α-helices. These structures are called EF- hand motifs as both α-helices are arranged like the thumb and the

forefinger of a hand. Fish parvalbumins have three EF-hand motifs (AB, CD, EF) but only the CD- and EF-motifs are functional and bind divalent cations (Ca2+, Mg2+). Parvalbumin structures rearrange globally upon ion-binding or release. The ion-binding capacity is essential for the correct parvalbumin function and its IgE-binding capability.<sup>430</sup> Parvalbumins with unfunctional calcium-binding sites bind IgE antibodies from fish-allergic patients only weakly.<sup>329</sup> Therefore, the functional calcium-binding motifs (CD, EF) have been suggested as important conformational B cell epitopes.

### Textbox Parvalbumin family characteristics

* Parvalbumins are members of the Ca2+-binding EF- hand superfamily.

* Clinical cross-reactivity is based on the presence of highly conserved IgE epitopes.

* Monosensitized patients have IgE to species-specific epitopes.

<table><caption>TABLE 105 Gad m 1 - homologus allergens from several fish and non-fish species</caption><thead><tr><td>Taxonomic order</td><td>Allergen source</td><td>Allergen</td></tr></thead><tbody><tr><td>Anura</td><td>Edible frog (Rana esculenta/Pelophylax esculentus)</td><td>Ran e 2*</td></tr><tr><td>Carcharhiniformes</td><td>Edible shark (Mustelus griseus)</td><td>Mus g 1</td></tr><tr><td rowspan="2">Clupeiformes</td><td>Atlantic herring (Clupea harengus)</td><td>Clu h 1*</td></tr><tr><td>Pacific pilchard (Sardinops sagax)</td><td>Sar sa 1*</td></tr><tr><td>Crocodylia</td><td>Australian saltwater crocodile (Crocodylus porosus)</td><td>Cro p 1*</td></tr><tr><td rowspan="2">Cypriniformes</td><td>Grass carp (Ctenopharyngodon idella)</td><td>Cten i 1*</td></tr><tr><td>Common carp (Cyprinus carpio)</td><td>Cyp c 1*</td></tr><tr><td rowspan="4">Gadiformes</td><td>Baltic cod ( Gadus callarias )</td><td>Gad c 1*</td></tr><tr><td>Atlantic cod ( Gadus morhua )</td><td>Gad m 1*</td></tr><tr><td>Atlantic hake ( Merluccius merluccius )</td><td>Mer mr 1</td></tr><tr><td>Alaska pollock ( Gadus chalcogrammus )</td><td>The c 1</td></tr><tr><td>Galliformes</td><td>Chicken ( Gallus gallus domesticus )</td><td>Gal d 8*</td></tr><tr><td rowspan="3">Perciformes</td><td>Barramundi ( Lates calcarifer )</td><td>Lat c 1*</td></tr><tr><td>Yellowfin tuna ( Thunnus albacares )</td><td>Thu a 1*</td></tr><tr><td>Swordfish ( Xiphias gladius )</td><td>Xip g 1*</td></tr><tr><td rowspan="2">Pleuronectiformes</td><td>Megrim ( Lepidorhombus whiffiagonis )</td><td>Lep w 1*</td></tr><tr><td>Common sole ( Solea solea )</td><td>Sole s 1*</td></tr><tr><td rowspan="2">Salmoniformes</td><td>Rainbow trout ( Onchorhynchus mykiss )</td><td>Onc m 1*</td></tr><tr><td>Atlantic salmon ( Salmo salar )</td><td>Sal s 1*</td></tr><tr><td rowspan="2">Scombriformes</td><td>Indian mackerel ( Rastrelliger kanagurta )</td><td>Ras k 1*</td></tr><tr><td>Atlantic mackerel ( Scomber scrombus )</td><td>Sco s 1*</td></tr><tr><td>Scorpaeniformes</td><td>Redfish ( Sebastes marinus )</td><td>Seb m 1*</td></tr><tr><td>Siluriformes</td><td>Striped catfish ( Pangasianodon hypophtalmus )</td><td>Pan h 1*</td></tr></tbody></table>

<footnote>* Allergens officially accepted and designated by the WHO/IUIS Allergen Nomenclature Sub-Committee (www.allergen.org); all others are unofficial names taken from the Allergome database (www.allergome.org).</footnote>

<footnote>* Allergens officially accepted and designated by the WHO/IUIS Allergen Nomenclature Sub-Committee (http://www.allergen.org); all others are unofficial names taken from the Allergome database (http://www.allergome.org).</footnote>
Allergens from the parvalbumin family have been identified and characterized in a number of fish.¹⁹⁵,¹⁰¹⁵ Homologs from 16 fish species have been validated and included in the official allergen nomenclature database (www.allergen.org, accessed 26 April 2022), but several other parvalbumins with IgE-binding properties can be found in other databases (Allergen Online, www.allergenonline.org; COMPARE, www.comparedatabase.org; Allergome, www.allergome.org). Detailed data on molecular and allergenic properties are available for parvalbumins from fish, which are commonly consumed in Europe such as cod, salmon, mackerel and tuna. Parvalbumin from cartilaginous fish (ray, shark), crocodile, frog and chicken, all mostly alpha-subtypes, are also included in the databases.¹⁰⁴⁵,¹⁶¹⁶,¹⁶⁷³ Some important representatives of the parvalbumin family are summarized in Table 105. Parvalbumins are small cytosolic molecules of 107-110 amino acids.¹⁰¹⁵ Several but most often two isoallergens can be found in the same fish muscle, as it was shown for salmon, cod and carp (www.allergen.org). These allergens were named beta1- and beta2-parvalbumins sharing a sequence identity of 64%, 72% and 84%, respectively.

Not all isoforms are necessarily included in the official allergen nomenclature database. Table 106 illustrates pairwise comparisons of amino acid sequence identities between parvalbumins from different species, which are commonly consumed in Europe. Highlighted in blue are the pairs of parvalbumin, which have been shown to be IgE cross-reactive in *in vitro* studies. This comparison shows that sequence identities vary over a broad range. However, IgE cross-reactivity has not been only reported for highly similar (98% identity) but even more distantly related fish parvalbumins (63% identity). This complies with the fact that the global protein structures are highly conserved and argues for common conformational B-cell epitopes.³²⁹

Fish parvalbumins are highly cross-reactive proteins; anti-parvalbumin IgE antibodies often recognize homologues from different fish supporting the fact that fish-allergic patients commonly react to multiple fish species (see chapter B12).¹⁰³⁶ While IgE cross-reactivity appears limited between alpha- and beta-parvalbumins, frequent cross-reactivities are observed among beta-homologues. The molecular basis for this high IgE cross-reactivity is the remarkable structural homology,

<figure><img src="image_313.png" /><figcaption>TABLE 106 Amino acid sequence identities (%) between parvalbumins registered by the WHO/IUIS Allergen Nomenclature Sub Committee</figcaption></figure>

The sequence identities were calculated using Multiple Sequence Alignment in Clustal Omega (EMBL-EBI, www.ebi.ac.uk/Tools/msa/clustalo/). Blue, documented IgE-cross-reactivity; bold, > 80% amino acid sequence identity; beta-1-isoform (in case of several isoallergens 1.0101) was used for comparison, except for the one highlighted with * which is alpha-parvalbumin
Allergens from the parvalbumin family have been identified and characterized in a number of fish.¹⁹⁵,¹⁰¹⁵ Homologs from 16 fish species have been validated and included in the official allergen nomenclature database (www.allergen.org, accessed 26 April 2022), but several other parvalbumins with IgE-binding properties can be found in other databases (Allergen Online, www.allergenonline.org; COMPARE, www.comparedatabase.org; Allergome, www.allergome.org). Detailed data on molecular and allergenic properties are available for parvalbumins from fish, which are commonly consumed in Europe such as cod, salmon, mackerel and tuna. Parvalbumin from cartilaginous fish (ray, shark), crocodile, frog and chicken, all mostly alpha-subtypes, are also included in the databases.¹⁰⁴⁵,¹⁶¹⁶,¹⁶⁷³ Some important representatives of the parvalbumin family are summarized in Table 105. Parvalbumins are small cytosolic molecules of 107-110 amino acids.¹⁰¹⁵ Several but most often two isoallergens can be found in the same fish muscle, as it was shown for salmon, cod and carp (www.allergen.org). These allergens were named beta1- and beta2-parvalbumins sharing a sequence identity of 64%, 72% and 84%, respectively.

Not all isoforms are necessarily included in the official allergen nomenclature database. Table 106 illustrates pairwise comparisons of amino acid sequence identities between parvalbumins from different species, which are commonly consumed in Europe. Highlighted in blue are the pairs of parvalbumin, which have been shown to be IgE cross-reactive in *in vitro* studies. This comparison shows that sequence identities vary over a broad range. However, IgE cross-reactivity has not been only reported for highly similar (98% identity) but even more distantly related fish parvalbumins (63% identity). This complies with the fact that the global protein structures are highly conserved and argues for common conformational B-cell epitopes.³²⁹

Fish parvalbumins are highly cross-reactive proteins; anti-parvalbumin IgE antibodies often recognize homologues from different fish supporting the fact that fish-allergic patients commonly react to multiple fish species (see chapter B12).¹⁰³⁶ While IgE cross-reactivity appears limited between alpha- and beta-parvalbumins, frequent cross-reactivities are observed among beta-homologues. The molecular basis for this high IgE cross-reactivity is the remarkable structural homology,

<figure><img src="image_321.png" /><figcaption>TABLE 106 Amino acid sequence identities (%) between parvalbumins registered by the WHO/IUIS Allergen Nomenclature Sub Committee</figcaption></figure>

The sequence identities were calculated using Multiple Sequence Alignment in Clustal Omega (EMBL-EBI, www.ebi.ac.uk/Tools/msa/clustalo/). Blue, documented IgE-cross-reactivity; bold, > 80% amino acid sequence identity; beta-1-isoform (in case of several isoallergens 1.0101) was used for comparison, except for the one highlighted with * which is alpha-parvalbumin
especially in the ion-binding regions.<sup>195,1015</sup> The surface comparison of selected cross-reactive fish allergens visualizes potential conformational B cell epitopes common to most parvalbumins (Figure 187).

However, a number of patients react only to specific or single fish. Tolerance of single species might be explained by very low allergen contents such as for tuna.<sup>1009</sup> Another reason for this clinical mono-/oligo-sensitivity are species-specific IgE-binding epitopes present on parvalbumins.<sup>1616</sup> It was also recently described that cartilaginous fish may be tolerated by patients sensitized to bony fish due to a low cross-reactivity between their only distantly related parvalbumins.<sup>208</sup> Studies on monosensitization to salmon/trout confirmed the presence of a salmonid-specific parvalbumin epitope, which is unique for these fish allergens<sup>1621</sup> (Figure 188).

Figure 189 represents documented and putative cross-reactivities among known fish and non-fish parvalbumins. During the past decade, new fish allergens have been characterized (see chapter B12). Future studies will have to address the characterization of selective marker molecules, parvalbumins and the new fish allergens, for IgE-based diagnosis to discriminate between patients with clinical cross-reactivity and fish species-specific sensitization.

The use of basophil activation assays with fish allergens appears to be promising and potentially exceeds the performance of simple IgE-binding assays.<sup>208,1617</sup> Their diagnostic relevance still needs to be further explored.

### 3 – Clinical relevance

Beta-parvalbumins are present in fish muscle and skin, but they become airborne upon handling and processing of fish.<sup>1015</sup> As such, they are both food and respiratory allergens but also potential contact allergens for occupationally exposed workers. Fish allergy is important in the domestic, public and occupational environment. Incidental episodes might occur upon inhalation of volatile allergens during fish preparation in the domestic environment. These allergens can be present as hidden allergens, for example, as contaminations of food that is not expected to contain fish and products thereof.<sup>558</sup> Respiratory problems of the upper and lower airway tract have been reported in the occupational context among workers processing fish.<sup>1052</sup> Both beta and alpha parvalbumins were shown to efficiently cross the epithelial barrier *in vitro*.<sup>1618</sup> A clear correlation has been

<figure><img src="image_314.png" /><figcaption>FIGURE 187 Surface comparisons of three different cross-reactive parvalbumins (based on PDB 2MBX, sequence conservation determined using the ConSurf tool). A. Cod Gad m 1, B. Salmon Sal s 1, C. Tuna Thu a 1; colored according to protein identity between Gad m 1 and the two others: blue, variable; white, average; purple/pink, conserved</figcaption></figure>

<figure><img src="image_315.png" /><figcaption>FIGURE 188 Surface comparisons of non-cross-reactive parvalbumins in salmonid-monosensitized patients (based on PDB 2MBX, sequence conservation determined using the ConSurf tool). A. Cod Gad m 1, B. Salmon Sal s 1, C. Trout Onc m 1. Yellow, Ca2+-binding sites; red, species-specific epitopes</figcaption></figure>
especially in the ion-binding regions.<sup>195,1015</sup> The surface comparison of selected cross-reactive fish allergens visualizes potential conformational B cell epitopes common to most parvalbumins (Figure 187).

However, a number of patients react only to specific or single fish. Tolerance of single species might be explained by very low allergen contents such as for tuna.<sup>1009</sup> Another reason for this clinical mono-/oligo-sensitivity are species-specific IgE-binding epitopes present on parvalbumins.<sup>1616</sup> It was also recently described that cartilaginous fish may be tolerated by patients sensitized to bony fish due to a low cross-reactivity between their only distantly related parvalbumins.<sup>208</sup> Studies on monosensitization to salmon/trout confirmed the presence of a salmonid-specific parvalbumin epitope, which is unique for these fish allergens<sup>1621</sup> (Figure 188).

Figure 189 represents documented and putative cross-reactivities among known fish and non-fish parvalbumins. During the past decade, new fish allergens have been characterized (see chapter B12). Future studies will have to address the characterization of selective marker molecules, parvalbumins and the new fish allergens, for IgE-based diagnosis to discriminate between patients with clinical cross-reactivity and fish species-specific sensitization.

The use of basophil activation assays with fish allergens appears to be promising and potentially exceeds the performance of simple IgE-binding assays.<sup>208,1617</sup> Their diagnostic relevance still needs to be further explored.

### 3 – Clinical relevance

Beta-parvalbumins are present in fish muscle and skin, but they become airborne upon handling and processing of fish.<sup>1015</sup> As such, they are both food and respiratory allergens but also potential contact allergens for occupationally exposed workers. Fish allergy is important in the domestic, public and occupational environment. Incidental episodes might occur upon inhalation of volatile allergens during fish preparation in the domestic environment. These allergens can be present as hidden allergens, for example, as contaminations of food that is not expected to contain fish and products thereof.<sup>558</sup> Respiratory problems of the upper and lower airway tract have been reported in the occupational context among workers processing fish.<sup>1052</sup> Both beta and alpha parvalbumins were shown to efficiently cross the epithelial barrier *in vitro*.<sup>1618</sup> A clear correlation has been

<figure><img src="image_322.png" /><figcaption>FIGURE 187 Surface comparisons of three different cross-reactive parvalbumins (based on PDB 2MBX, sequence conservation determined using the ConSurf tool). A. Cod Gad m 1, B. Salmon Sal s 1, C. Tuna Thu a 1; colored according to protein identity between Gad m 1 and the two others: blue, variable; white, average; purple/pink, conserved</figcaption></figure>

<figure><img src="image_323.png" /><figcaption>FIGURE 188 Surface comparisons of non-cross-reactive parvalbumins in salmonid-monosensitized patients (based on PDB 2MBX, sequence conservation determined using the ConSurf tool). A. Cod Gad m 1, B. Salmon Sal s 1, C. Trout Onc m 1. Yellow, Ca2+-binding sites; red, species-specific epitopes</figcaption></figure>
<figure><img src="image_316.png" /><figcaption>FIGURE 189 Cross-reactivities among allergenic fish and non-fish parvalbumins. All parvalbumins have a high potential for cross-reactivity based on high sequence homology. Lines represent documented IgE-cross-reactivity. Clockwise: Atlantic cod, Atlantic mackerel, megrim, common sole, Atlantic salmon, rainbow trout, Atlantic hake, crocodile, chicken, edible frog, Alaska pollock, yellowfin tuna, common carp, Atlantic herring</figcaption></figure>

shown for the development of work-related asthma and fish allergy as explained by the high environmental fish allergen concentrations in the workplace.

Studies estimated that <1% of the general population suffers from an allergy to fish (see chapter B12).³²⁹ Children often maintain their clinical allergy to fish during adolescence.⁹⁷⁶ However, a recent study reported that the tolerance of fish increases from childhood into adulthood, with about half of the fish-allergic children reaching fish tolerance in adolescence.¹⁰³⁵ Fish allergy is more frequent in countries with large coastal regions characterized by frequent fish consumption and settling of fish-processing industries. Concerning the prevalence of specific IgE to parvalbumins in fish-allergic patients, it has been stated for a long time that more than 90% are sensitized to this pan-allergen. According to results of more recent studies, this prevalence seems to be considerably lower (see chapter B12). However, parvalbumin appears to still be the major allergen.¹⁹⁵

The main route of sensitization to fish parvalbumins is thought to be by ingestion, thus by uptake through the gastrointestinal tract. Epidermal or airway sensitization might also play a role, especially in children with active atopic dermatitis at the time of solid food introduction into diet¹⁰³⁵ and through inhalation of airborne allergens in occupational settings.¹⁰¹⁴ Common clinical manifestations include mild (oral allergy syndrome, erythema) to moderate (urticaria, vomiting, diarrhea) or severe (angioedema,

bronchospasm, anaphylaxis) symptoms.¹⁹⁵,¹⁰¹⁵ Sensitized patients on antacid medication are at higher risk to develop severe reactions than others.¹⁶¹⁹

A key feature of potent food allergens is their stability to thermal treatments. Fish parvalbumins are extremely heat stable, and therefore, they are still detectable in products processed by cooking or frying or in pickled food.¹⁰⁰⁹ This emphasizes their undiminished allergenicity upon various food preparation methods. Fish parvalbumins become glycosylated by heating in the presence of glucose through the Maillard reaction. However, the resulting effects on their allergenic potency, be it cumulative or diminishing, still require further investigations.

### Textbox Clinical relevance

* Parvalbumins are fish pan-allergens.

* Parvalbumins are food, respiratory and contact allergens.

* Beta-parvalbumins retain IgE-binding properties upon food processing.

* Low cross-reactivity between beta-parvalbumins from bony fish and alpha-parvalbumins from cartilaginous fish has been observed.
<figure><img src="image_324.png" /><figcaption>FIGURE 189 Cross-reactivities among allergenic fish and non-fish parvalbumins. All parvalbumins have a high potential for cross-reactivity based on high sequence homology. Lines represent documented IgE-cross-reactivity. Clockwise: Atlantic cod, Atlantic mackerel, megrim, common sole, Atlantic salmon, rainbow trout, Atlantic hake, crocodile, chicken, edible frog, Alaska pollock, yellowfin tuna, common carp, Atlantic herring</figcaption></figure>

shown for the development of work-related asthma and fish allergy as explained by the high environmental fish allergen concentrations in the workplace.

Studies estimated that <1% of the general population suffers from an allergy to fish (see chapter B12).³²⁹ Children often maintain their clinical allergy to fish during adolescence.⁹⁷⁶ However, a recent study reported that the tolerance of fish increases from childhood into adulthood, with about half of the fish-allergic children reaching fish tolerance in adolescence.¹⁰³⁵ Fish allergy is more frequent in countries with large coastal regions characterized by frequent fish consumption and settling of fish-processing industries. Concerning the prevalence of specific IgE to parvalbumins in fish-allergic patients, it has been stated for a long time that more than 90% are sensitized to this pan-allergen. According to results of more recent studies, this prevalence seems to be considerably lower (see chapter B12). However, parvalbumin appears to still be the major allergen.¹⁹⁵

The main route of sensitization to fish parvalbumins is thought to be by ingestion, thus by uptake through the gastrointestinal tract. Epidermal or airway sensitization might also play a role, especially in children with active atopic dermatitis at the time of solid food introduction into diet¹⁰³⁵ and through inhalation of airborne allergens in occupational settings.¹⁰¹⁴ Common clinical manifestations include mild (oral allergy syndrome, erythema) to moderate (urticaria, vomiting, diarrhea) or severe (angioedema,

bronchospasm, anaphylaxis) symptoms.¹⁹⁵,¹⁰¹⁵ Sensitized patients on antacid medication are at higher risk to develop severe reactions than others.¹⁶¹⁹

A key feature of potent food allergens is their stability to thermal treatments. Fish parvalbumins are extremely heat stable, and therefore, they are still detectable in products processed by cooking or frying or in pickled food.¹⁰⁰⁹ This emphasizes their undiminished allergenicity upon various food preparation methods. Fish parvalbumins become glycosylated by heating in the presence of glucose through the Maillard reaction. However, the resulting effects on their allergenic potency, be it cumulative or diminishing, still require further investigations.

### Textbox Clinical relevance

* Parvalbumins are fish pan-allergens.

* Parvalbumins are food, respiratory and contact allergens.

* Beta-parvalbumins retain IgE-binding properties upon food processing.

* Low cross-reactivity between beta-parvalbumins from bony fish and alpha-parvalbumins from cartilaginous fish has been observed.
Currently, there is no causal therapy available to treat allergic sensitization to fish parvalbumins. The therapeutic desensitization with increasing doses of fish is disadvised in clinical routine practice, as the risk for anaphylactic reactions cannot be ruled out. Often, a strict avoidance diet is recommended—except for selectively tolerated species (see chapter B12). Caution is advised with products of fish origin, which might be contaminated with parvalbumins or contain other fish allergens (fish collagen and gelatin). An important perspective for the future treatment of fish allergy could be the development of hypoallergenic low IgE-binding parvalbumins,⁴³⁰ (see Chapter A09). Beyond, novel procedures in fish farming, tailored to reduce the allergenicity of fish parvalbumin, might be another prospective approach.¹⁶²⁰

## 4 - Clinical management

The mainstay for the diagnosis of fish allergy are the record of the patient's medical history, the analysis of the skin prick test reactivity using fish extracts or the dorsal-rostral part of selected fish or the potentially symptoms-eliciting food source, the quantification of serum IgE antibodies, and in some cases, oral food challenges (see chapter “Fish allergy”). Commercial IgE-quantification assays have long been available from Thermo Fisher Scientific (ImmunoCAP Specific IgE test and ImmunoCAP ISAC, www.thermofisher.com) for about 30 extracts from different fish species as well as two recombinant parvalbumins, Gad c 1 from cod and Cyp c 1 from carp. A recently developed ALEX2 multiplex platform (MacroArray Diagnostics, www.macroarraydx.com) made available a number of parvalbumins and total extracts from several fish species, along with other fish allergens such as cod enolase and cod aldolase (6 different fish species plus 9 fish allergens).

Highly sensitized patients often react to various fish. First, they should be cautiously tested, due to potential reactions, for skin reactivity to cod, salmon and the symptoms-eliciting fish, followed by analysis of specific IgE binding to cod and salmon extract. Second, a polysensitization to fish can be confirmed by detecting specific IgE to the cross-reactive parvalbumin from cod (Gad m 1). Future IgE testing for other allergens will entail a more specific diagnosis

of these patients. With the advent of novel IgE-multiplexing platforms, the testing of sensitization to parvalbumins from distantly related bony fish as well as cartilaginous fish such as ray, is possible. Indeed, if this IgE testing in parvalbumin-positive patients is negative (Figure 190), there is a high probability that ray will be tolerated,²⁰⁸ which needs to be confirmed by oral provocation.

### Textbox Advices

* Perform skin testing with cod and salmon muscle.

* Be aware that a false negative result might be obtained with dark fish muscle!

* Test specific IgE to cod and salmon extract.

* Test specific IgE to purified cod parvalbumin.

However, a number of fish-allergic individuals react to specific fish only.¹⁰²⁴,¹⁰³⁵ The challenge of future studies will be the identification of marker allergens for IgE tests to discriminate between these poly- and oligo-/ mono-sensitized patients. As for now, the discriminative significance of anti-parvalbumin IgE antibodies seems to be limited because they are often cross-reactive with various homologues, which does not necessarily imply a clinical reactivity. An exception has been reported for a subgroup of patients with mono-sensitivity to salmonid fish. They might be diagnosed efficiently by determination of specific IgE to salmon and trout parvalbumin (see “Clinical case 2”).¹⁶²¹ However, the positive predictive value (PPV) for single parvalbumins in the diagnosis of allergy to single fish still needs to be defined.

Overall, allergens different from parvalbumins might be elicitors for fish allergy. Cod enolases and aldolases have been identified as responsible allergens in three clinical cases of mono-sensitivity to cod (see Chapter B12).¹⁰³⁷ Even other allergens such as fish gelatin, fish collagen and others might play a role in this context (Figure 191)¹⁰¹²,¹⁰²⁵ Anyway, diagnostic conclusions from *in vitro* IgE results, especially negative results, should be confirmed by further oral provocation tests in case of a strong suspicion of a fish allergy.

<figure><img src="image_317.png" /><figcaption>FIGURE 190 Added value of the use of single allergens in the case of a positive IgE test to cod extract</figcaption></figure>
Currently, there is no causal therapy available to treat allergic sensitization to fish parvalbumins. The therapeutic desensitization with increasing doses of fish is disadvised in clinical routine practice, as the risk for anaphylactic reactions cannot be ruled out. Often, a strict avoidance diet is recommended—except for selectively tolerated species (see chapter B12). Caution is advised with products of fish origin, which might be contaminated with parvalbumins or contain other fish allergens (fish collagen and gelatin). An important perspective for the future treatment of fish allergy could be the development of hypoallergenic low IgE-binding parvalbumins,⁴³⁰ (see Chapter A09). Beyond, novel procedures in fish farming, tailored to reduce the allergenicity of fish parvalbumin, might be another prospective approach.¹⁶²⁰

## 4 - Clinical management

The mainstay for the diagnosis of fish allergy are the record of the patient's medical history, the analysis of the skin prick test reactivity using fish extracts or the dorsal-rostral part of selected fish or the potentially symptoms-eliciting food source, the quantification of serum IgE antibodies, and in some cases, oral food challenges (see chapter “Fish allergy”). Commercial IgE-quantification assays have long been available from Thermo Fisher Scientific (ImmunoCAP Specific IgE test and ImmunoCAP ISAC, www.thermofisher.com) for about 30 extracts from different fish species as well as two recombinant parvalbumins, Gad c 1 from cod and Cyp c 1 from carp. A recently developed ALEX2 multiplex platform (MacroArray Diagnostics, www.macroarraydx.com) made available a number of parvalbumins and total extracts from several fish species, along with other fish allergens such as cod enolase and cod aldolase (6 different fish species plus 9 fish allergens).

Highly sensitized patients often react to various fish. First, they should be cautiously tested, due to potential reactions, for skin reactivity to cod, salmon and the symptoms-eliciting fish, followed by analysis of specific IgE binding to cod and salmon extract. Second, a polysensitization to fish can be confirmed by detecting specific IgE to the cross-reactive parvalbumin from cod (Gad m 1). Future IgE testing for other allergens will entail a more specific diagnosis

of these patients. With the advent of novel IgE-multiplexing platforms, the testing of sensitization to parvalbumins from distantly related bony fish as well as cartilaginous fish such as ray, is possible. Indeed, if this IgE testing in parvalbumin-positive patients is negative (Figure 190), there is a high probability that ray will be tolerated,²⁰⁸ which needs to be confirmed by oral provocation.

### Textbox Advices

* Perform skin testing with cod and salmon muscle.

* Be aware that a false negative result might be obtained with dark fish muscle!

* Test specific IgE to cod and salmon extract.

* Test specific IgE to purified cod parvalbumin.

However, a number of fish-allergic individuals react to specific fish only.¹⁰²⁴,¹⁰³⁵ The challenge of future studies will be the identification of marker allergens for IgE tests to discriminate between these poly- and oligo-/ mono-sensitized patients. As for now, the discriminative significance of anti-parvalbumin IgE antibodies seems to be limited because they are often cross-reactive with various homologues, which does not necessarily imply a clinical reactivity. An exception has been reported for a subgroup of patients with mono-sensitivity to salmonid fish. They might be diagnosed efficiently by determination of specific IgE to salmon and trout parvalbumin (see “Clinical case 2”).¹⁶²¹ However, the positive predictive value (PPV) for single parvalbumins in the diagnosis of allergy to single fish still needs to be defined.

Overall, allergens different from parvalbumins might be elicitors for fish allergy. Cod enolases and aldolases have been identified as responsible allergens in three clinical cases of mono-sensitivity to cod (see Chapter B12).¹⁰³⁷ Even other allergens such as fish gelatin, fish collagen and others might play a role in this context (Figure 191)¹⁰¹²,¹⁰²⁵ Anyway, diagnostic conclusions from *in vitro* IgE results, especially negative results, should be confirmed by further oral provocation tests in case of a strong suspicion of a fish allergy.

<figure><img src="image_325.png" /><figcaption>FIGURE 190 Added value of the use of single allergens in the case of a positive IgE test to cod extract</figcaption></figure>
## 5 - Clinical cases

In the clinical cases presented in this paragraph, the following parvalbumins have been included in IgE-based diagnostic procedures during research studies: salmon Sal s 1, carp Cyp c 1, cod Gad m 1, tuna Thu a 1, trout Onc m 1, crocodile Cro p 1.

**Case 1** (published¹⁰¹⁹)

**Clinical History:** A male child, 12 years old, with a clinical history of fish allergy since early childhood presenting with angioedema and respiratory problems upon ingestion of different fish as well as with acute urticaria when touching fish.

**Test with extracts:** Skin tests performed with commercial extracts were positive for cod, salmon and tuna. IgE to cod, salmon and tuna extract were positive (16 kU/L, 32 kU/L and 65 kU/L, respectively).

**Food challenge:** The parents of the child refused a food challenge.

**Test with molecules:** Cod, salmon and tuna parvalbumins were positive in IgE ELISA (20 kU/L, 18 kU/L and 30 kU/L, respectively).

**Conclusion:** The polysensitization to multiple fish species was confirmed in this case by revealing cross-reactive IgE antibodies to homologue parvalbumins from different species. (Figure 192)

**Case 2** (published¹⁶²¹)

**Clinical History:** A female patient, 21 years old, with a clinical history of fish allergy since childhood presenting with swelling of the tongue and oral mucosa, facial edema and vomiting minutes after ingestion of salmon or trout.

**Test with extracts:** Skin tests performed with commercial extracts were positive for salmon and trout but negative for other fish. Only IgE to salmon extract was slightly positive (0.4 kU/L).

**Food challenge:** As the patient repeatedly experienced symptoms with salmonid fish, she refused to be tested by oral provocation.

**Test with molecules:** As determined by IgE ELISA, salmon and trout parvalbumins were positive at 0.2-0.4 kU/L. No inhibition assays were performed with other fish parvalbumins as IgE binding was negative for cod, carp, mackerel, redfish and herring homologues.

**Conclusion:** In this case, clinical species-specific sensitivity to salmonid fish was confirmed by specific IgE to salmon and trout parvalbumin (Figure 193).

<figure><img src="image_318.png" /><figcaption>FIGURE 191 Added value of the use of single allergens in the case of a positive IgE test to tuna extract</figcaption></figure>

<figure><img src="image_319.png" /><figcaption>FIGURE 192 Diagnostic procedure for case 1</figcaption></figure>

<figure><img src="image_320.png" /><figcaption>FIGURE 193 Diagnostic procedure for case 2</figcaption></figure>
## 5 - Clinical cases

In the clinical cases presented in this paragraph, the following parvalbumins have been included in IgE-based diagnostic procedures during research studies: salmon Sal s 1, carp Cyp c 1, cod Gad m 1, tuna Thu a 1, trout Onc m 1, crocodile Cro p 1.

**Case 1** (published¹⁰¹⁹)

**Clinical History:** A male child, 12 years old, with a clinical history of fish allergy since early childhood presenting with angioedema and respiratory problems upon ingestion of different fish as well as with acute urticaria when touching fish.

**Test with extracts:** Skin tests performed with commercial extracts were positive for cod, salmon and tuna. IgE to cod, salmon and tuna extract were positive (16 kU/L, 32 kU/L and 65 kU/L, respectively).

**Food challenge:** The parents of the child refused a food challenge.

**Test with molecules:** Cod, salmon and tuna parvalbumins were positive in IgE ELISA (20 kU/L, 18 kU/L and 30 kU/L, respectively).

**Conclusion:** The polysensitization to multiple fish species was confirmed in this case by revealing cross-reactive IgE antibodies to homologue parvalbumins from different species. (Figure 192)

**Case 2** (published¹⁶²¹)

**Clinical History:** A female patient, 21 years old, with a clinical history of fish allergy since childhood presenting with swelling of the tongue and oral mucosa, facial edema and vomiting minutes after ingestion of salmon or trout.

**Test with extracts:** Skin tests performed with commercial extracts were positive for salmon and trout but negative for other fish. Only IgE to salmon extract was slightly positive (0.4 kU/L).

**Food challenge:** As the patient repeatedly experienced symptoms with salmonid fish, she refused to be tested by oral provocation.

**Test with molecules:** As determined by IgE ELISA, salmon and trout parvalbumins were positive at 0.2-0.4 kU/L. No inhibition assays were performed with other fish parvalbumins as IgE binding was negative for cod, carp, mackerel, redfish and herring homologues.

**Conclusion:** In this case, clinical species-specific sensitivity to salmonid fish was confirmed by specific IgE to salmon and trout parvalbumin (Figure 193).

<figure><img src="image_326.png" /><figcaption>FIGURE 191 Added value of the use of single allergens in the case of a positive IgE test to tuna extract</figcaption></figure>

<figure><img src="image_327.png" /><figcaption>FIGURE 192 Diagnostic procedure for case 1</figcaption></figure>

<figure><img src="image_328.png" /><figcaption>FIGURE 193 Diagnostic procedure for case 2</figcaption></figure>
<figure><img src="image_321.png" /><figcaption>FIGURE 194 Diagnostic procedure for case 3</figcaption></figure>

**Case 3** (published<sup>1622</sup>)

**Clinical History:** Male patient, 9 years old, presenting with intense oral itching, perioral erythema, dyspnea, and generalized urticaria within minutes of eating a small portion of crocodile burger for the first time.

**Test with extracts:** Skin tests with commercial extracts were positive for most fish (cod, salmon, trout, tuna, anchovy, megrim, sole, hake, anglerfish, sardine) but negative for swordfish. Prick-to-prick was positive with raw and cooked crocodile meat, raw conger body and cooked conger body.

Specific IgE was positive for most fish (cod 100, salmon 68, trout 20.6, tuna 2.7, megrim 8.1, sole 41.7, hake 15.4, sardine 5.4, swordfish 1.3 kU/L).

**Food challenge:** No oral food challenge was performed.

**Test with molecules:** IgE binding was detected for cod parvalbumin (r Gad c 1, 24.6 kU/L) and in immunoblot, for crocodile parvalbumin Cro p 1.

**Conclusion:** An anaphylactic reaction to crocodile meat was explained by primary sensitization to fish parvalbumin with cross-reactivity to the crocodile homologue (Figure 194).
<figure><img src="image_329.png" /><figcaption>FIGURE 194 Diagnostic procedure for case 3</figcaption></figure>

**Case 3** (published<sup>1622</sup>)

**Clinical History:** Male patient, 9 years old, presenting with intense oral itching, perioral erythema, dyspnea, and generalized urticaria within minutes of eating a small portion of crocodile burger for the first time.

**Test with extracts:** Skin tests with commercial extracts were positive for most fish (cod, salmon, trout, tuna, anchovy, megrim, sole, hake, anglerfish, sardine) but negative for swordfish. Prick-to-prick was positive with raw and cooked crocodile meat, raw conger body and cooked conger body.

Specific IgE was positive for most fish (cod 100, salmon 68, trout 20.6, tuna 2.7, megrim 8.1, sole 41.7, hake 15.4, sardine 5.4, swordfish 1.3 kU/L).

**Food challenge:** No oral food challenge was performed.

**Test with molecules:** IgE binding was detected for cod parvalbumin (r Gad c 1, 24.6 kU/L) and in immunoblot, for crocodile parvalbumin Cro p 1.

**Conclusion:** An anaphylactic reaction to crocodile meat was explained by primary sensitization to fish parvalbumin with cross-reactivity to the crocodile homologue (Figure 194).
Conflict of Interest - MAUG 2.0

S.D. is the Chair of EAACI Scientific Media Communications Committee (til 2022/07) and reports research grants from Euroimmun Labordiagnostika, consulting fees from Leti Pharma, Bencard Allergie GmbH and OMRON Healthcare Technologies Co. Ltd and has received speaker's honoraria from Bencard Allergie GmbH; and Allergopharma and is in the advisory board from Bencard Allergie GmbH.

C.H. is the Chair of Allergy Diagnosis and Systems Medicine Interest Group of EAACI and receives grants from Fonds National de la Recherche, Luxembourg and has a research collaboration with MacroArray Diagnostics; received speaker's honorarium from ALK and travel support from HAL Allergy.

A.F.S. is Chair of the EAACI interest group Food Allergy and has received grants from MRC UK, NIH USA, FARE USA; Asthma UK, Rosetrees Trust, Wellcome Trust, and BBSRC; reports consulting fees from IgGenix, Novartis, Allergy Therapeutics; and Stallergenes; Speaker's honoraria from Novartis, ThermoFisher, Nutricia, Buhlmann, and Nestle and is in the advisory board from Allergy Therapeutics;

N.A. received grant support from the Ministry of Science of the Republic of Colombia (Grants 756-2017, 806-2018) and reports speaker's honorarium from Sanofi at scientific events;

F.A. reports royalties: from Eleva GmbH for patent "fucosyl-transferase" (1999);

B.B-W. reports consulting fees from ALK, Allergopharma, Menarini, Sanofi, Novartis, and Thermofisher; speaker's honoraria from ALK, Allergopharma, Menarini, Sanofi, Novartis, and Thermofisher and is a member of the advisory board from Aimmune;

D.B. reports consulting fees from ALK A/S;

K.B. is board member of: AGATE (Anaphylaxis Training and Education); GPA (German Society for Pediatric Allergology & Environmental Medicine); DGKJ (German Society for Allergology & Clinical Immunology) and DAAB (German Allergy and Asthma Association). She holds research grants from Aimmune, ALK, Danone, DBV, Hipp, Hycor, and Infectopharm; reports consulting fees from Hipp and speaker's honoraria from Aimmune, Danone, Infectopharm, Mylan/meda, Nestle, Novartis, and ThermoFisher; she is a member of the advisory board from Aimmune, Bencard, Danone, DBV, Hycor, Jenapharma, Mylan/Meda, Nestle, and Novartis;

T.B. is the President German Society of Dermatology and reports grants from German Research Foundation (SFB 1371 TP 06; BI 696/12-1); AbbVie, ALK-Abello, Celgene-BMS, and the Lilly, Novartis, Sanofi, Regeneron, Viatris; consulting fees from ALK-Abello, Lilly, Novartis, and Sanofi; speaker's honoraria from AbbVie, ALK-Abello, Lilly, Novartis, Sanofi, Regeneron; and is a member of the advisory board from LEO Pharma, Lilly, and Sanofi;

S.B. received grants from Thermo Fisher Sci., Bencard Allergie GmbH, Allergy Therapeutics plc, and Leti Pharma; consulting fees from Bencard Allergie GmbH and speaker honoraria from DGAKI, Bencard; Allergie GmbH, and Thermo Fisher Sci.

H.B. reports grant support from Austrian Science Fund (FWF), Danube Allergy Research Cluster funded by the Country of Lower Austria; author Royalties from UpToDate Inc. (Wolters Kluwer

Health); and lecturer's honorarium from Imperial College London, "Allergy Master of Sciences Programme"

He.Bro. is board member of BSACI Paediatric Allergy Group and President Elect of the Royal Society of Medicine; She reports grants from NIH Seal Study and Aimmune; royalties from Wiley Rapid Paediatrics and Speaker's honoraria from DBV Technologies, Sanofi, and GSK; travel support from EAACI, BSACI; and is in the advisory board of SEED Study and IREACH Study;

M.D.C. is co-owner, shareholder and employee of Indoor Biotechnologies;

M.C. reports grant support from R01 NIH grant with INDOOR Biotechnologies;

A.C. is board member of WAO and the British Society of Allergy and Clinical Immunology; he received a grant from MRC (UK): MR/S025340/1; reports consulting fees from Philipps, Worg Pharmaceuticals, Stallergenes-Greer; speaker's honoraria from AstraZeneca, Novartis, and GSK;

R.C. reports grants from Australian NHMRC Aus Pollen Partnership (GNT1116107); Abionic SA (Switzerland) co-funding of collaboration research grant from NFMRI; and patent: US PTO 14/311944 owned by QUT; AU2008/316301 owned by QUT;

A.M.E. reports grants from Nutricia Research, Utrecht, The Netherlands; and Euroimmun AG, Lübeck, Germany;

P.A.E. reports grant support from Ulrich Muller Gierock Foundation; consulting fees from DBV, Nestle, Danone, Novartis, Abbott, Alexeia and ThermoFisher; travel support from EAACI, ThermoFisher; royalties from upToDate; Elsevier; speaker's honoraria from TF, Abbott; ALK; and holds stock options from DBV Technologies;

F.G.P. receives grant support from the Span. Ministry of Economics and Competition: PI17/01318, AC18/00031; RETICS ARADyAL (RD16/0006/0001, RD16/0006/0007); Sen. Clin. Res. Program B-0005-2019;

C.Ha. reports grant support from Danube Allergy Research Cluster funded by Country of Lower Austria; and is a member of the advisory board from Allergios;

R.G. H. is supported by the Johns Hopkins University School of Medicine faculty Scholarship;

T.H. reports speaker's honoraria from ALK, Bencard, Takeda ALK, Novartis, and HAL; travel support from Novartis; and is in the advisory board from Novartis, Bencard, ALK, and Takeda;

H.J.H. is the current treasurer of the Danish Society of Allergology; he reports grant from the Danish Innovation Funds; and is filing Patent 74490PC01; has received travel support from AAAAI;

T.Ho. is advisory board member from Deutscher Allergie- und Asthmabund eV, DAAB; German Federal Office of Consumer Protection and Food Safety; Senate Commission on Food Safety of the German Research Foundation, DFG-SKLM; International Life Sciences Institute, ILSI Europe; received grants from European Dir. for the Quality of Medicines and Health Care; EU-COST Action ImpARAS; German Federal Ministry of Food and Agriculture; and obtained travel support from Food Allergy Forum April, 2019, Bastiaanse-communication, Bilthoven, NL;
Conflict of Interest - MAUG 2.0

S.D. is the Chair of EAACI Scientific Media Communications Committee (til 2022/07) and reports research grants from Euroimmun Labordiagnostika, consulting fees from Leti Pharma, Bencard Allergie GmbH and OMRON Healthcare Technologies Co. Ltd and has received speaker's honoraria from Bencard Allergie GmbH; and Allergopharma and is in the advisory board from Bencard Allergie GmbH.

C.H. is the Chair of Allergy Diagnosis and Systems Medicine Interest Group of EAACI and receives grants from Fonds National de la Recherche, Luxembourg and has a research collaboration with MacroArray Diagnostics; received speaker's honorarium from ALK and travel support from HAL Allergy.

A.F.S. is Chair of the EAACI interest group Food Allergy and has received grants from MRC UK, NIH USA, FARE USA; Asthma UK, Rosetrees Trust, Wellcome Trust, and BBSRC; reports consulting fees from IgGenix, Novartis, Allergy Therapeutics; and Stallergenes; Speaker's honoraria from Novartis, ThermoFisher, Nutricia, Buhlmann, and Nestle and is in the advisory board from Allergy Therapeutics;

N.A. received grant support from the Ministry of Science of the Republic of Colombia (Grants 756-2017, 806-2018) and reports speaker's honorarium from Sanofi at scientific events;

F.A. reports royalties: from Eleva GmbH for patent "fucosyl-transferase" (1999);

B.B-W. reports consulting fees from ALK, Allergopharma, Menarini, Sanofi, Novartis, and Thermofisher; speaker's honoraria from ALK, Allergopharma, Menarini, Sanofi, Novartis, and Thermofisher and is a member of the advisory board from Aimmune;

D.B. reports consulting fees from ALK A/S;

K.B. is board member of: AGATE (Anaphylaxis Training and Education); GPA (German Society for Pediatric Allergology & Environmental Medicine); DGKJ (German Society for Allergology & Clinical Immunology) and DAAB (German Allergy and Asthma Association). She holds research grants from Aimmune, ALK, Danone, DBV, Hipp, Hycor, and Infectopharm; reports consulting fees from Hipp and speaker's honoraria from Aimmune, Danone, Infectopharm, Mylan/meda, Nestle, Novartis, and ThermoFisher; she is a member of the advisory board from Aimmune, Bencard, Danone, DBV, Hycor, Jenapharma, Mylan/Meda, Nestle, and Novartis;

T.B. is the President German Society of Dermatology and reports grants from German Research Foundation (SFB 1371 TP 06; BI 696/12-1); AbbVie, ALK-Abello, Celgene-BMS, and the Lilly, Novartis, Sanofi, Regeneron, Viatris; consulting fees from ALK-Abello, Lilly, Novartis, and Sanofi; speaker's honoraria from AbbVie, ALK-Abello, Lilly, Novartis, Sanofi, Regeneron; and is a member of the advisory board from LEO Pharma, Lilly, and Sanofi;

S.B. received grants from Thermo Fisher Sci., Bencard Allergie GmbH, Allergy Therapeutics plc, and Leti Pharma; consulting fees from Bencard Allergie GmbH and speaker honoraria from DGAKI, Bencard; Allergie GmbH, and Thermo Fisher Sci.

H.B. reports grant support from Austrian Science Fund (FWF), Danube Allergy Research Cluster funded by the Country of Lower Austria; author Royalties from UpToDate Inc. (Wolters Kluwer

Health); and lecturer's honorarium from Imperial College London, "Allergy Master of Sciences Programme"

He.Bro. is board member of BSACI Paediatric Allergy Group and President Elect of the Royal Society of Medicine; She reports grants from NIH Seal Study and Aimmune; royalties from Wiley Rapid Paediatrics and Speaker's honoraria from DBV Technologies, Sanofi, and GSK; travel support from EAACI, BSACI; and is in the advisory board of SEED Study and IREACH Study;

M.D.C. is co-owner, shareholder and employee of Indoor Biotechnologies;

M.C. reports grant support from R01 NIH grant with INDOOR Biotechnologies;

A.C. is board member of WAO and the British Society of Allergy and Clinical Immunology; he received a grant from MRC (UK): MR/S025340/1; reports consulting fees from Philipps, Worg Pharmaceuticals, Stallergenes-Greer; speaker's honoraria from AstraZeneca, Novartis, and GSK;

R.C. reports grants from Australian NHMRC Aus Pollen Partnership (GNT1116107); Abionic SA (Switzerland) co-funding of collaboration research grant from NFMRI; and patent: US PTO 14/311944 owned by QUT; AU2008/316301 owned by QUT;

A.M.E. reports grants from Nutricia Research, Utrecht, The Netherlands; and Euroimmun AG, Lübeck, Germany;

P.A.E. reports grant support from Ulrich Muller Gierock Foundation; consulting fees from DBV, Nestle, Danone, Novartis, Abbott, Alexeia and ThermoFisher; travel support from EAACI, ThermoFisher; royalties from upToDate; Elsevier; speaker's honoraria from TF, Abbott; ALK; and holds stock options from DBV Technologies;

F.G.P. receives grant support from the Span. Ministry of Economics and Competition: PI17/01318, AC18/00031; RETICS ARADyAL (RD16/0006/0001, RD16/0006/0007); Sen. Clin. Res. Program B-0005-2019;

C.Ha. reports grant support from Danube Allergy Research Cluster funded by Country of Lower Austria; and is a member of the advisory board from Allergios;

R.G. H. is supported by the Johns Hopkins University School of Medicine faculty Scholarship;

T.H. reports speaker's honoraria from ALK, Bencard, Takeda ALK, Novartis, and HAL; travel support from Novartis; and is in the advisory board from Novartis, Bencard, ALK, and Takeda;

H.J.H. is the current treasurer of the Danish Society of Allergology; he reports grant from the Danish Innovation Funds; and is filing Patent 74490PC01; has received travel support from AAAAI;

T.Ho. is advisory board member from Deutscher Allergie- und Asthmabund eV, DAAB; German Federal Office of Consumer Protection and Food Safety; Senate Commission on Food Safety of the German Research Foundation, DFG-SKLM; International Life Sciences Institute, ILSI Europe; received grants from European Dir. for the Quality of Medicines and Health Care; EU-COST Action ImpARAS; German Federal Ministry of Food and Agriculture; and obtained travel support from Food Allergy Forum April, 2019, Bastiaanse-communication, Bilthoven, NL;
T.J. is board member of the German Society of Allergy and Clinical Immunology; he has received grants from: ALK Abello, Novartis; speaker's honoraria from ALK Abello, Novartis, Thermo Fisher Sci., and Allergy Therapeutics/Bencard; he is in the advisory board from ALK Abello, Novartis, and Bencard;

U.J. is member of the Scientific Board of the German Society for Allergology and Clinical Immunology (DGAKI); Executive Committee of the WHO/IUIS Allergen Nomenclature Subcommittee; editorial Board Member of the Journal "Allergy." She reports grants from the German Research Foundation: DFG JA 1007/2-3; Federal Ministry of Education and Science: DZL: German Center for Lung Research; AA-FPB; FKZ:01EA2109B; Verbundprojekt ImprovNgDlagnostiCs and Therapy; INDICATE-FH BMEL and BLE AptaSens; has received consulting fees from Frauenhofer Zukunftsstiftung; and files for Patent: Project 14-00142 (Basophil-Activation Test), pending;

M.J. is EAACI President (til 2022/07); reports consulting fees from Allergopharma, Stallergenes, Regeneron, Pfizer, and Chiesi; speaker's honoraria from Stallergenes Greer, Allergopharma, and HAL Allergy; is in the advisory board from Allergopharma and receives clinical Investigator honoraria from GSK, AstraZeneca, Regeneron, Genentech, Takeda, Chiesi, Novartis, Allergopharma, Stallergenes, Allergy Therapeutics, HAL Allergy, ALK Abello, Shire, Celltrion, and Verona Pharma;

J.K-T. is a member of the German Society of Allergy and Clinical Immunology; reports grants from GSK, Lofarma, Novartis, Regeneron, and Stallergenes-Greer; received honoraria from AllergenOnline (Nebraska, USA), Allergy Therapeutics, Allergopharma, ALK-Abelló, AstraZeneca, Bencard, HAL Allergy, LETI, Sanofi, Springer International Publishers, ThermoFisher Sci, and Thieme Publishers, Germany;

E.K. is EAACI Board member and received speaker's honoraria and travel support from ThermoFisher Sci.

J.K. reports speaker's honoraria from ThermoFisher Sci.

G.L. Grants: NIAID, NIH, FARE, Action Medical Research; MRC & Asthma UK Centre, UK Dept of Health through NIHR, National Peanut Board, The Davis Foundation; Consulting fees: DBV, Novartis; ALK Abello, Lurie Children's Hospital; speakers honoraria: DBV, ALK Abello, EPG Health; scientific advisory board member: DBV Technologies; National Peanut Board; Lurie Children's Hospital; stock holder: DBV Technology; shareholder: Mission Mighty Me;

T-M.L. reports speaker's honoraria from ThermoFisher Sci.

O.L. reports consulting fees received from ThermoFisher Sci.; speaker's honoraria from ThermoFisher Sci.; GSK, AstraZeneca; Sanofi, Novartis; WAO; and Vall d'Hebron Research Institute; and travel support from: Sanofi; she is a member of the advisory board from Sanofi and AstraZeneca;

C.M. is nonexecutive Director from Reacta Biotech Ltd until November 2019; Member of the Food Standards Agency Advisory Committee on Novel Foods and Processes; and holds founder shares of Reacta Biotech Ltd; she reports grants from Reacta B UK Research and Innovation - Innovate, Swindon, UK, Biotech Ltd, European Food Safety Authority, Food Standards Agency, London, UK; consultancy fees received from Reacta Biotech Ltd; travel support from Romer Laboratories UK Ltd to attend CIFSQ, Shanghai, China, Dec 2019; and is patent holder Int. Patent Application (PCT); Oral Food

Challenge Meal Formulations-Kit for allergy diagnosis. Application number:PCT/GB2016/051637; Int. Patent application (PCT): Oral Food Challenge Meal Formulations: Kits comprising placebo and nonplacebo oral food challenge meal formulations. Application number: PCT/GB2016/053829;

M.M. is a member of the Food allergy working group of the French Society of Allergology; reports grant support from Novartis; received speaker's honoraria from Stallergenes Greer, Novartis, ALK, and ThermoFisher;

A.M. is GA2LEN Executive Committee member; she reports grants obtained from Aimmune, DVB Technologies, and Novartis; speaker's honoraria from Aimmune, DVB Technologies, Nestlè Health Science, Nestlè Purina, and Viatris; and is member of the advisory board from Viatris, Novartis, Aimmune, DVB technologies; and member of the Data safety monitoring board from Regeneron;

A. N-W. is committee member of IFPIES, Annals of Allergy, Asthma and Immunology, and American Board of Allergy and Immunology; received grant support from DBV Technologies, Regeneron, Alladapt; and consulting fees from Aquestive; speaker's honoraria from Nestle, Danone, and ThermoFisher Sci;

M.O. is Vice president of Science EAACI; he reports grant support from Fonds National de la Recherche Luxembourg, consulting fees from Hycor Biomedical; speaker's honoraria from Hycor Biomedical, ThermoFisher Sci. Phadia, consulting fees from the Ministry of Health, Government of Luxembourg; travel support from EAACI for annual congresses; patents pending: WO2019/076478AI; WO2019/076477AI; scientific co-founder of Tolerogenics SARL, Esch-sur-Alzette;

S.P. received grants from National Institutes of Health, NIAID; Food Allergy Science Initiative; and filing of patent: Monoclonal antibody (pending);

A.P. is employed by InBio Inc; she reports grant from NIH Award Number R01AI077653 (to AP); received speaker's honorarium from ThermoFisher Sci.

E.P. reports grant support from Hycor Biomedical;

L.K.P. is board member of the Asthma and Allergy Denmark (Patient organization) and Research Council, CIA (Collegium International Allergologicum); advisory Board Member of COMPARE Allergen Database HESI; reports consulting fees from Novozymes A/S and speaker's honoraria from HVD Holding AG Poland;

C.R. receives grant from the Austrian Science Fund (FWF);

S.R. reports grant support from ITN070AD, (Impact of Ad Libitum Peanut Consumption and Genetics in LEAP Participants and Families: LEAP Participant, Sibling and Parent Cohorts (LEAP Trio),

M.R. is executive board member of the German Society for Allergology and Clinical Immunology (DGAKI); member of EAACI interest group IG Envir./Occup. Allergy and member of several task forces; Editor of the Journal "Allergologie/Allergologie Select; received speaker's honoraria from ThermoFisher Sci. and Leti;

J.S. is committee member of SEAIC; EAACI, and AAAI; reports grant support from Sanofi; and consulting fees from Sanofi, ABBVie; Novartis; speaker's honoraria from Sanofi, GSK, FAES Farma; and travel support from Sanofi;
T.J. is board member of the German Society of Allergy and Clinical Immunology; he has received grants from: ALK Abello, Novartis; speaker's honoraria from ALK Abello, Novartis, Thermo Fisher Sci., and Allergy Therapeutics/Bencard; he is in the advisory board from ALK Abello, Novartis, and Bencard;

U.J. is member of the Scientific Board of the German Society for Allergology and Clinical Immunology (DGAKI); Executive Committee of the WHO/IUIS Allergen Nomenclature Subcommittee; editorial Board Member of the Journal "Allergy." She reports grants from the German Research Foundation: DFG JA 1007/2-3; Federal Ministry of Education and Science: DZL: German Center for Lung Research; AA-FPB; FKZ:01EA2109B; Verbundprojekt ImprovNgDlagnostiCs and Therapy; INDICATE-FH BMEL and BLE AptaSens; has received consulting fees from Frauenhofer Zukunftsstiftung; and files for Patent: Project 14-00142 (Basophil-Activation Test), pending;

M.J. is EAACI President (til 2022/07); reports consulting fees from Allergopharma, Stallergenes, Regeneron, Pfizer, and Chiesi; speaker's honoraria from Stallergenes Greer, Allergopharma, and HAL Allergy; is in the advisory board from Allergopharma and receives clinical Investigator honoraria from GSK, AstraZeneca, Regeneron, Genentech, Takeda, Chiesi, Novartis, Allergopharma, Stallergenes, Allergy Therapeutics, HAL Allergy, ALK Abello, Shire, Celltrion, and Verona Pharma;

J.K-T. is a member of the German Society of Allergy and Clinical Immunology; reports grants from GSK, Lofarma, Novartis, Regeneron, and Stallergenes-Greer; received honoraria from AllergenOnline (Nebraska, USA), Allergy Therapeutics, Allergopharma, ALK-Abelló, AstraZeneca, Bencard, HAL Allergy, LETI, Sanofi, Springer International Publishers, ThermoFisher Sci, and Thieme Publishers, Germany;

E.K. is EAACI Board member and received speaker's honoraria and travel support from ThermoFisher Sci.

J.K. reports speaker's honoraria from ThermoFisher Sci.

G.L. Grants: NIAID, NIH, FARE, Action Medical Research; MRC & Asthma UK Centre, UK Dept of Health through NIHR, National Peanut Board, The Davis Foundation; Consulting fees: DBV, Novartis; ALK Abello, Lurie Children's Hospital; speakers honoraria: DBV, ALK Abello, EPG Health; scientific advisory board member: DBV Technologies; National Peanut Board; Lurie Children's Hospital; stock holder: DBV Technology; shareholder: Mission Mighty Me;

T-M.L. reports speaker's honoraria from ThermoFisher Sci.

O.L. reports consulting fees received from ThermoFisher Sci.; speaker's honoraria from ThermoFisher Sci.; GSK, AstraZeneca; Sanofi, Novartis; WAO; and Vall d'Hebron Research Institute; and travel support from: Sanofi; she is a member of the advisory board from Sanofi and AstraZeneca;

C.M. is nonexecutive Director from Reacta Biotech Ltd until November 2019; Member of the Food Standards Agency Advisory Committee on Novel Foods and Processes; and holds founder shares of Reacta Biotech Ltd; she reports grants from Reacta B UK Research and Innovation - Innovate, Swindon, UK, Biotech Ltd, European Food Safety Authority, Food Standards Agency, London, UK; consultancy fees received from Reacta Biotech Ltd; travel support from Romer Laboratories UK Ltd to attend CIFSQ, Shanghai, China, Dec 2019; and is patent holder Int. Patent Application (PCT); Oral Food

Challenge Meal Formulations-Kit for allergy diagnosis. Application number:PCT/GB2016/051637; Int. Patent application (PCT): Oral Food Challenge Meal Formulations: Kits comprising placebo and nonplacebo oral food challenge meal formulations. Application number: PCT/GB2016/053829;

M.M. is a member of the Food allergy working group of the French Society of Allergology; reports grant support from Novartis; received speaker's honoraria from Stallergenes Greer, Novartis, ALK, and ThermoFisher;

A.M. is GA2LEN Executive Committee member; she reports grants obtained from Aimmune, DVB Technologies, and Novartis; speaker's honoraria from Aimmune, DVB Technologies, Nestlè Health Science, Nestlè Purina, and Viatris; and is member of the advisory board from Viatris, Novartis, Aimmune, DVB technologies; and member of the Data safety monitoring board from Regeneron;

A. N-W. is committee member of IFPIES, Annals of Allergy, Asthma and Immunology, and American Board of Allergy and Immunology; received grant support from DBV Technologies, Regeneron, Alladapt; and consulting fees from Aquestive; speaker's honoraria from Nestle, Danone, and ThermoFisher Sci;

M.O. is Vice president of Science EAACI; he reports grant support from Fonds National de la Recherche Luxembourg, consulting fees from Hycor Biomedical; speaker's honoraria from Hycor Biomedical, ThermoFisher Sci. Phadia, consulting fees from the Ministry of Health, Government of Luxembourg; travel support from EAACI for annual congresses; patents pending: WO2019/076478AI; WO2019/076477AI; scientific co-founder of Tolerogenics SARL, Esch-sur-Alzette;

S.P. received grants from National Institutes of Health, NIAID; Food Allergy Science Initiative; and filing of patent: Monoclonal antibody (pending);

A.P. is employed by InBio Inc; she reports grant from NIH Award Number R01AI077653 (to AP); received speaker's honorarium from ThermoFisher Sci.

E.P. reports grant support from Hycor Biomedical;

L.K.P. is board member of the Asthma and Allergy Denmark (Patient organization) and Research Council, CIA (Collegium International Allergologicum); advisory Board Member of COMPARE Allergen Database HESI; reports consulting fees from Novozymes A/S and speaker's honoraria from HVD Holding AG Poland;

C.R. receives grant from the Austrian Science Fund (FWF);

S.R. reports grant support from ITN070AD, (Impact of Ad Libitum Peanut Consumption and Genetics in LEAP Participants and Families: LEAP Participant, Sibling and Parent Cohorts (LEAP Trio),

M.R. is executive board member of the German Society for Allergology and Clinical Immunology (DGAKI); member of EAACI interest group IG Envir./Occup. Allergy and member of several task forces; Editor of the Journal "Allergologie/Allergologie Select; received speaker's honoraria from ThermoFisher Sci. and Leti;

J.S. is committee member of SEAIC; EAACI, and AAAI; reports grant support from Sanofi; and consulting fees from Sanofi, ABBVie; Novartis; speaker's honoraria from Sanofi, GSK, FAES Farma; and travel support from Sanofi;
S.S. received speaker's honorarium from Mylan EPD;

E.S. reports consulting fees and speaker's honoraria from ThermoFisher and Stallergenes;

P. S-G. is treasurer of the Int. Society for Atopic Dermatitis; reports support from Christine Kühne Center for Allergy Research and Education CK-CARE; speaker's honorarium from ThermoFisher; and research collaboration with ThermoFisher, Buehlmann, and Euroimmune.

M.V-M. is advisory board member of Allergy Therapeutics; received speaker's honorarium from GSK; and consulting fees from Organon;

M.vH. received speaker's honorarium from ThermoFisher Sci

R.vR. board member of HESI Protein Allergenicity and Toxins Bioinformatics Committee; holds stock options for Anagany Inc; received grants from Dutch Science Foundation; Health Holland; Angany Inc.; consulting fees from HAL Allergy BV; Citeq Inc., Anagany Inc., Reacta Healthcare Ltd, Mission MightyMe, and AB Enzymes; speaker's honoraria from ThermoFisher Sci. HAL Allergy BV and ALK; travel support from Anagany Inc.

M.W. received consulting fees from DBV, Nestle, Danone, Novartis, Abbott, and Alexeia;

J.Z. reports grant support (Grant 803-2018);

P.M.M. reports grants from Deutsche Forschungsgesellschaft DFG: MA47/2-1; consulting fees from Hycor Biomedical; Euroimmune; ThermoFisher; and speaker's Honorarium from Hycor Biomedical;

K.H-S. is EAACI board member (til 2022/07), receives grant from Danube Allergy Research Cluster funded by Country of Lower Austria; consulting fees for COMPARE allergen database; and speaker's honorarium from EMMA Mtd;

All other authors have no COI to declare.

ORCID

Stephanie Dramburg https://orcid.org/0000-0002-9303-3260

Christiane Hilger https://orcid.org/0000-0001-9455-5442

Alexandra F. Santos https://orcid.org/0000-0002-7805-1436

Leticia de las Vecillas https://orcid.org/0000-0003-4969-5678

Rob C. Aalberse https://orcid.org/0000-0002-3902-6596

Nathalie Acevedo https://orcid.org/0000-0002-5154-2964

Lorenz Aglas https://orcid.org/0000-0002-1236-5934

Friedrich Altmann https://orcid.org/0000-0002-0112-7877

Karla L. Arruda https://orcid.org/0000-0002-7505-210X

Riccardo Asero https://orcid.org/0000-0002-8277-1700

Barbara Ballmer-Weber https://orcid.org/0000-0002-4136-5036

Domingo Barber https://orcid.org/0000-0002-5488-5700

Kirsten Beyer https://orcid.org/0000-0003-1859-0419

Tilo Biedermann https://orcid.org/0000-0002-5352-5105

Maria Beatrice Bilo https://orcid.org/0000-0002-9324-6039

Simon Blank https://orcid.org/0000-0002-5649-1078

Philipp P. Bosshard https://orcid.org/0000-0002-1154-2281

Heimo Breiteneder https://orcid.org/0000-0003-2022-8689

Helen A. Brough https://orcid.org/0000-0001-7203-0813

Merima Bublin https://orcid.org/0000-0002-7191-9431

Dianne Campbell https://orcid.org/0000-0002-0907-6963

Luis Caraballo https://orcid.org/0000-0002-5204-1109

Jean Christoph Caubet https://orcid.org/0000-0001-5006-5724

Martin D. Chapman https://orcid.org/0000-0002-0845-3632

Maksymilian Chruszcz https://orcid.org/0000-0001-7521-5485

Adnan Custovic https://orcid.org/0000-0001-5218-7071

Rebecca Czolk https://orcid.org/0000-0003-4296-3051

Janet Davies https://orcid.org/0000-0002-6378-4119

Nikolaos Douladiris https://orcid.org/0000-0003-4037-4421

Bernadette Eberlein https://orcid.org/0000-0003-4509-6491

Motohiro Ebisawa https://orcid.org/0000-0003-4117-558X

Anna Ehlers https://orcid.org/0000-0001-7833-2900

Philippe Eigenmann https://orcid.org/0000-0003-1738-1826

Gabriele Gadermaier https://orcid.org/0000-0002-4886-417X

Mattia Giovannini https://orcid.org/0000-0001-9568-6882

Francisca Gomez https://orcid.org/0000-0003-1607-6686

Carole Guillet https://orcid.org/0000-0003-3809-0526

Christine Hafner https://orcid.org/0000-0003-3745-1414

Michael Hauser https://orcid.org/0000-0001-6558-755X

Hans Jürgen Hoffmann https://orcid.org/0000-0002-6743-7931

Thomas Holzhauser https://orcid.org/0000-0002-7818-7261

Tomona Lizuka https://orcid.org/0000-0002-1946-277X

Alain Jacquet https://orcid.org/0000-0002-0980-9741

Thilo Jakob https://orcid.org/0000-0002-3124-9998

Bente Janssen-Weets https://orcid.org/0000-0003-3379-7792

Uta Jappe https://orcid.org/0000-0001-5602-3672

Tanja Kalic https://orcid.org/0000-0002-9641-0244

Sandip Kamath https://orcid.org/0000-0002-5956-8552

Sabine Kespohl https://orcid.org/0000-0001-7571-1323

Edward Knol https://orcid.org/0000-0001-7368-9820

André Knulst https://orcid.org/0000-0002-1056-3179

Jon R. Konradsen https://orcid.org/0000-0001-7745-8624

Peter Korošec https://orcid.org/0000-0002-0835-1599

Annette Kuehn https://orcid.org/0000-0003-0474-2135

Gideon Lack https://orcid.org/0000-0001-7350-4021

Thuy-My Le https://orcid.org/0000-0001-6571-220X

Andreas Lopata https://orcid.org/0000-0002-2940-9235

Olga Luengo https://orcid.org/0000-0002-8905-2776

Clare Mills https://orcid.org/0000-0001-7433-1740

Martine Morisset https://orcid.org/0000-0002-4028-703X

Antonella Muraro https://orcid.org/0000-0002-5026-5862

Anna Nowak-Wegrzyn https://orcid.org/0000-0002-0960-9854

Roni Nugraha https://orcid.org/0000-0001-5935-5867

Markus Ollert https://orcid.org/0000-0002-8055-0103

Kati Palosuo https://orcid.org/0000-0002-4357-0439

Elide Anna Pastorello https://orcid.org/0000-0002-3753-1783

Sarita Ulhas Patil https://orcid.org/0000-0002-2172-2286

Thomas Platts-Mills https://orcid.org/0000-0002-1263-329X

Anna Pomés https://orcid.org/0000-0002-8729-1829

Pascal Poncet https://orcid.org/0000-0003-0757-5424

Ekaterina Potapova https://orcid.org/0000-0003-4427-6335

Lars K. Poulsen https://orcid.org/0000-0002-1730-847X

Christian Radauer https://orcid.org/0000-0001-9920-4449
S.S. received speaker's honorarium from Mylan EPD;

E.S. reports consulting fees and speaker's honoraria from ThermoFisher and Stallergenes;

P. S-G. is treasurer of the Int. Society for Atopic Dermatitis; reports support from Christine Kühne Center for Allergy Research and Education CK-CARE; speaker's honorarium from ThermoFisher; and research collaboration with ThermoFisher, Buehlmann, and Euroimmune.

M.V-M. is advisory board member of Allergy Therapeutics; received speaker's honorarium from GSK; and consulting fees from Organon;

M.vH. received speaker's honorarium from ThermoFisher Sci

R.vR. board member of HESI Protein Allergenicity and Toxins Bioinformatics Committee; holds stock options for Anagany Inc; received grants from Dutch Science Foundation; Health Holland; Angany Inc.; consulting fees from HAL Allergy BV; Citeq Inc., Anagany Inc., Reacta Healthcare Ltd, Mission MightyMe, and AB Enzymes; speaker's honoraria from ThermoFisher Sci. HAL Allergy BV and ALK; travel support from Anagany Inc.

M.W. received consulting fees from DBV, Nestle, Danone, Novartis, Abbott, and Alexeia;

J.Z. reports grant support (Grant 803-2018);

P.M.M. reports grants from Deutsche Forschungsgesellschaft DFG: MA47/2-1; consulting fees from Hycor Biomedical; Euroimmune; ThermoFisher; and speaker's Honorarium from Hycor Biomedical;

K.H-S. is EAACI board member (til 2022/07), receives grant from Danube Allergy Research Cluster funded by Country of Lower Austria; consulting fees for COMPARE allergen database; and speaker's honorarium from EMMA Mtd;

All other authors have no COI to declare.

ORCID

Stephanie Dramburg https://orcid.org/0000-0002-9303-3260

Christiane Hilger https://orcid.org/0000-0001-9455-5442

Alexandra F. Santos https://orcid.org/0000-0002-7805-1436

Leticia de las Vecillas https://orcid.org/0000-0003-4969-5678

Rob C. Aalberse https://orcid.org/0000-0002-3902-6596

Nathalie Acevedo https://orcid.org/0000-0002-5154-2964

Lorenz Aglas https://orcid.org/0000-0002-1236-5934

Friedrich Altmann https://orcid.org/0000-0002-0112-7877

Karla L. Arruda https://orcid.org/0000-0002-7505-210X

Riccardo Asero https://orcid.org/0000-0002-8277-1700

Barbara Ballmer-Weber https://orcid.org/0000-0002-4136-5036

Domingo Barber https://orcid.org/0000-0002-5488-5700

Kirsten Beyer https://orcid.org/0000-0003-1859-0419

Tilo Biedermann https://orcid.org/0000-0002-5352-5105

Maria Beatrice Bilo https://orcid.org/0000-0002-9324-6039

Simon Blank https://orcid.org/0000-0002-5649-1078

Philipp P. Bosshard https://orcid.org/0000-0002-1154-2281

Heimo Breiteneder https://orcid.org/0000-0003-2022-8689

Helen A. Brough https://orcid.org/0000-0001-7203-0813

Merima Bublin https://orcid.org/0000-0002-7191-9431

Dianne Campbell https://orcid.org/0000-0002-0907-6963

Luis Caraballo https://orcid.org/0000-0002-5204-1109

Jean Christoph Caubet https://orcid.org/0000-0001-5006-5724

Martin D. Chapman https://orcid.org/0000-0002-0845-3632

Maksymilian Chruszcz https://orcid.org/0000-0001-7521-5485

Adnan Custovic https://orcid.org/0000-0001-5218-7071

Rebecca Czolk https://orcid.org/0000-0003-4296-3051

Janet Davies https://orcid.org/0000-0002-6378-4119

Nikolaos Douladiris https://orcid.org/0000-0003-4037-4421

Bernadette Eberlein https://orcid.org/0000-0003-4509-6491

Motohiro Ebisawa https://orcid.org/0000-0003-4117-558X

Anna Ehlers https://orcid.org/0000-0001-7833-2900

Philippe Eigenmann https://orcid.org/0000-0003-1738-1826

Gabriele Gadermaier https://orcid.org/0000-0002-4886-417X

Mattia Giovannini https://orcid.org/0000-0001-9568-6882

Francisca Gomez https://orcid.org/0000-0003-1607-6686

Carole Guillet https://orcid.org/0000-0003-3809-0526

Christine Hafner https://orcid.org/0000-0003-3745-1414

Michael Hauser https://orcid.org/0000-0001-6558-755X

Hans Jürgen Hoffmann https://orcid.org/0000-0002-6743-7931

Thomas Holzhauser https://orcid.org/0000-0002-7818-7261

Tomona lizuka https://orcid.org/0000-0002-1946-277X

Alain Jacquet https://orcid.org/0000-0002-0980-9741

Thilo Jakob https://orcid.org/0000-0002-3124-9998

Bente Janssen-Weets https://orcid.org/0000-0003-3379-7792

Uta Jappe https://orcid.org/0000-0001-5602-3672

Tanja Kalic https://orcid.org/0000-0002-9641-0244

Sandip Kamath https://orcid.org/0000-0002-5956-8552

Sabine Kespohl https://orcid.org/0000-0001-7571-1323

Edward Knol https://orcid.org/0000-0001-7368-9820

André Knulst https://orcid.org/0000-0002-1056-3179

Jon R. Konradsen https://orcid.org/0000-0001-7745-8624

Peter Korošec https://orcid.org/0000-0002-0835-1599

Annette Kuehn https://orcid.org/0000-0003-0474-2135

Gideon Lack https://orcid.org/0000-0001-7350-4021

Thuy-My Le https://orcid.org/0000-0001-6571-220X

Andreas Lopata https://orcid.org/0000-0002-2940-9235

Olga Luengo https://orcid.org/0000-0002-8905-2776

Clare Mills https://orcid.org/0000-0001-7433-1740

Martine Morisset https://orcid.org/0000-0002-4028-703X

Antonella Muraro https://orcid.org/0000-0002-5026-5862

Anna Nowak-Wegrzyn https://orcid.org/0000-0002-0960-9854

Roni Nugraha https://orcid.org/0000-0001-5935-5867

Markus Ollert https://orcid.org/0000-0002-8055-0103

Kati Palosuo https://orcid.org/0000-0002-4357-0439

Elide Anna Pastorello https://orcid.org/0000-0002-3753-1783

Sarita Ulhas Patil https://orcid.org/0000-0002-2172-2286

Thomas Platts-Mills https://orcid.org/0000-0002-1263-329X

Anna Pomés https://orcid.org/0000-0002-8729-1829

Pascal Poncet https://orcid.org/0000-0003-0757-5424

Ekaterina Potapova https://orcid.org/0000-0003-4427-6335

Lars K. Poulsen https://orcid.org/0000-0002-1730-847X

Christian Radauer https://orcid.org/0000-0001-9920-4449
Suzana Radulovic https://orcid.org/0000-0003-1154-8365

Monika Raulf https://orcid.org/0000-0001-6315-120X

Pierre Rougé https://orcid.org/0000-0002-9936-7843

Joaquin Sastre https://orcid.org/0000-0003-4689-6837

Sakura Sato https://orcid.org/0000-0003-3674-0759

Enrico Scala https://orcid.org/0000-0002-9391-9168

Johannes M. Schmid https://orcid.org/0000-0002-4558-2943

Peter Schmid-Grendelmeier https://orcid.org/0000-0003-3215-3370

Denise Schrama https://orcid.org/0000-0003-0274-5957

Hélène Sénéchal https://orcid.org/0000-0002-8415-8803

Claudia Traidl-Hoffmann https://orcid.org/0000-0001-5085-5179

Marcela Valverde-Monge https://orcid.org/0000-0002-0172-3951

Marianne van Hage https://orcid.org/0000-0003-3091-1596

Ronald van Ree https://orcid.org/0000-0003-0767-0894

Kitty Verhoeckx https://orcid.org/0000-0002-6557-3198

Stefan Vieths https://orcid.org/0000-0003-3826-6704

Magnus Wickman https://orcid.org/0000-0003-2710-8620

Josefina Zakzuk https://orcid.org/0000-0002-6007-5376

Paolo M. Matricardi https://orcid.org/0000-0001-5485-0324

Karin Hoffmann-Sommergruber https://orcid.org/0000-0002-8830-058X

REFERENCES

1. Ishizaka K, Ishizaka T. Identification of gamma-E-antibodies as a carrier of reaginic activity. *J Immunol*. 1967;99(6):1187-1198.

2. WHO/IUIS Allergen Nomenclature Home Page. Accessed September 4, 2021. http://allergen.org/

3. IUIS/WHO Allergen Nomenclature Subcommittee. Allergen nomenclature. IUIS/WHO Allergen Nomenclature Subcommittee. *Bull World Health Organ*. 1994;72(5):797-806.

4. Matricardi PM, Kleine-Tebbe J, Hoffmann HJ, et al. EAACI molecular allergology user's guide. *Pediatr Allergy Immunol*. 2016;27(Suppl 23):1-250.

5. Johnson P, Marsh DG. 'Isoallergens' from rye grass pollen. *Nature*. 1965;206(987):935-937.

6. King TP, Norman PS, Connell JT. Isolation and characterization of allergens from ragweed pollen. *II. Biochemistry*. 1964;3:458-468.

7. Aas K, Jebsen JW. Studies of hypersensitivity to fish. Partial purification and crystallization of a major allergenic component of cod. *Int Arch Allergy Appl Immunol*. 1967;32(1):1-20.

8. Chapman MD, Platts-Mills TA. Purification and characterization of the major allergen from Dermatophagoides pteronyssinus-antigen P1. *J Immunol*. 1980;125(2):587-592.

9. Radauer C, Nandy A, Ferreira F, et al. Update of the WHO/IUIS Allergen Nomenclature Database based on analysis of allergen sequences. *Allergy*. 2014;69(4):413-419.

10. King TP, Hoffman D, Löwenstein H, Marsh DG, Platts-Mills TA, Thomas W. Allergen nomenclature. *Allergy*. 1995;50(9):765-774.

11. Ackaert C, Kofler S, Horejs-Hoeck J, et al. The impact of nitration on the structure and immunogenicity of the major birch pollen allergen Bet v 1.0101. *PLoS One*. 2014;9(8):e104520.

12. Aalberse RC. Recombinant allergens need a reality check. *J Allergy Clin Immunol*. 2022;149(3):901-903.

13. Aalberse RC. Structural biology of allergens. *J Allergy Clin Immunol*. 2000;106(2):228-238.

14. Aalberse RC, Akkerdaas J, van Ree R. Cross-reactivity of IgE antibodies to allergens. *Allergy*. 2001;56(6):478-490.

15. Pascal M, Grishina G, Yang AC, et al. Molecular diagnosis of shrimp allergy: efficiency of several allergens to predict clinical reactivity. *J Allergy Clin Immunol Pract*. 2015;3(4):521-529.

16. Aalberse RC, Koshte V, Clemens JG. Immunoglobulin E antibodies that crossreact with vegetable foods, pollen, and Hymenoptera venom. *J Allergy Clin Immunol*. 1981;68(5):356-364.

17. Platts-Mills TA, Hilger C, Jappe U, et al. Carbohydrate epitopes currently recognized as targets for IgE antibodies. *Allergy*. 2021;76(8):2383-2394.

18. Platts-Mills TA, Schuyler AJ, Tripathi A, Commins SP. Anaphylaxis to the carbohydrate side chain alpha-gal. *Immunol Allergy Clin North Am*. 2015;35(2):247-260.

19. van Ree R. Carbohydrate epitopes and their relevance for the diagnosis and treatment of allergic diseases. *Int Arch Allergy Immunol*. 2002;129(3):189-197.

20. Platts-Mills TAE, Li RC, Keshavarz B, Smith AR, Wilson JM. Diagnosis and management of patients with the α-Gal syndrome. *J Allergy Clin Immunol Pract*. 2020;8(1):15-23.

21. Murangi T, Prakash P, Moreira BP, et al. Ascaris lumbricoides and ticks associated with sensitization to galactose α1,3-galactose and elicitation of the alpha-gal syndrome. *J Allergy Clin Immunol*. 2022;149(2):698-707.

22. Aalberse RC, Crameri R. IgE-binding epitopes: a reappraisal. *Allergy*. 2011;66(10):1261-1274.

23. Grüber C, Lau S, Dannemann A, Sommerfeld C, Wahn U, Aalberse RC. Down-regulation of IgE and IgG4 antibodies to tetanus toxoid and diphtheria toxoid by covaccination with cellular Bordetella pertussis vaccine. *J Immunol*. 2001;167(4):2411-2417.

24. Caraballo L, Valenta R, Acevedo N, Zakzuk J. Are the terms major and minor allergens useful for precision allergology? *Front Immunol*. 2021;12:651500.

25. Breiteneder H, Mills EN. Molecular properties of food allergens. *J Allergy Clin Immunol*. 2005;115(1):14-23; quiz 24.

26. Virtanen T, Kinnunen T, Rytkönen-Nissinen M. Mammalian lipocalin allergens—insights into their enigmatic allergenicity. *Clin Exp Allergy*. 2012;42(4):494-504.

27. Westerhout J, Krone T, Snippe A, et al. Allergicenicity prediction of novel and modified proteins: not a mission impossible! Development of a Random Forest allergicenicity prediction model. *Regul Toxicol Pharmacol*. 2019;107:104422.

28. Posa D, Perna S, Resch Y, et al. Evolution and predictive value of IgE responses toward a comprehensive panel of house dust mite allergens during the first 2 decades of life. *J Allergy Clin Immunol*. 2017;139(2):541-549.

29. Sarzinszky E, Lupinek C, Vrtala S, et al. Expression in *Escherichia coli* and purification of folded rDer p 20, the arginine kinase from dermatophagoides pteronyssinus: a possible biomarker for allergic asthma. *Allergy Asthma Immunol Res*. 2021;13(1):154-163.

30. Siroux V, Boudier A, Nadif R, Lupinek C, Valenta R, Bousquet J. Association between asthma, rhinitis, and conjunctivitis morbidities with molecular IgE sensitization in adults. *Allergy*. 2019;74(4):824-827.

31. Nicolaou N, Poorafshar M, Murray C, et al. Allergy or tolerance in children sensitized to peanut: prevalence and differentiation using component-resolved diagnostics. *J Allergy Clin Immunol*. 2010;125(1):191-197.

32. Vieths S, Scheurer S, Ballmer-Weber B. Current understanding of cross-reactivity of food allergens and pollen. *Ann N Y Acad Sci*. 2002;964:47-68.

33. Sancho Al, Wangorsch A, Jensen BM, et al. Responsiveness of the major birch allergen Bet v 1 scaffold to the gastric environment: impact on structure and allergenic activity. *Mol Nutr Food Res*. 2011;55(11):1690-1699.

34. Reuter A, Lidholm J, Andersson K, et al. A critical assessment of allergen component-based in vitro diagnosis in cherry allergy across Europe. *Clin Exp Allergy*. 2006;36(6):815-823.
Suzana Radulovic https://orcid.org/0000-0003-1154-8365

Monika Raulf https://orcid.org/0000-0001-6315-120X

Pierre Rougé https://orcid.org/0000-0002-9936-7843

Joaquin Sastre https://orcid.org/0000-0003-4689-6837

Sakura Sato https://orcid.org/0000-0003-3674-0759

Enrico Scala https://orcid.org/0000-0002-9391-9168

Johannes M. Schmid https://orcid.org/0000-0002-4558-2943

Peter Schmid-Grendelmeier https://orcid.org/0000-0003-3215-3370

Denise Schrama https://orcid.org/0000-0003-0274-5957

Hélène Sénéchal https://orcid.org/0000-0002-8415-8803

Claudia Traidl-Hoffmann https://orcid.org/0000-0001-5085-5179

Marcela Valverde-Monge https://orcid.org/0000-0002-0172-3951

Marianne van Hage https://orcid.org/0000-0003-3091-1596

Ronald van Ree https://orcid.org/0000-0003-0767-0894

Kitty Verhoeckx https://orcid.org/0000-0002-6557-3198

Stefan Vieths https://orcid.org/0000-0003-3826-6704

Magnus Wickman https://orcid.org/0000-0003-2710-8620

Josefina Zakzuk https://orcid.org/0000-0002-6007-5376

Paolo M. Matricardi https://orcid.org/0000-0001-5485-0324

Karin Hoffmann-Sommergruber https://orcid.org/0000-0002-8830-058X

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2. WHO/IUIS Allergen Nomenclature Home Page. Accessed September 4, 2021. http://allergen.org/

3. IUIS/WHO Allergen Nomenclature Subcommittee. Allergen nomenclature. IUIS/WHO Allergen Nomenclature Subcommittee. *Bull World Health Organ*. 1994;72(5):797-806.

4. Matricardi PM, Kleine-Tebbe J, Hoffmann HJ, et al. EAACI molecular allergology user's guide. *Pediatr Allergy Immunol*. 2016;27(Suppl 23):1-250.

5. Johnson P, Marsh DG. 'Isoallergens' from rye grass pollen. *Nature*. 1965;206(987):935-937.

6. King TP, Norman PS, Connell JT. Isolation and characterization of allergens from ragweed pollen. *II. Biochemistry*. 1964;3:458-468.

7. Aas K, Jebsen JW. Studies of hypersensitivity to fish. Partial purification and crystallization of a major allergenic component of cod. *Int Arch Allergy Appl Immunol*. 1967;32(1):1-20.

8. Chapman MD, Platts-Mills TA. Purification and characterization of the major allergen from Dermatophagoides pteronyssinus-antigen P1. *J Immunol*. 1980;125(2):587-592.

9. Radauer C, Nandy A, Ferreira F, et al. Update of the WHO/IUIS Allergen Nomenclature Database based on analysis of allergen sequences. *Allergy*. 2014;69(4):413-419.

10. King TP, Hoffman D, Löwenstein H, Marsh DG, Platts-Mills TA, Thomas W. Allergen nomenclature. *Allergy*. 1995;50(9):765-774.

11. Ackaert C, Kofler S, Horejs-Hoeck J, et al. The impact of nitration on the structure and immunogenicity of the major birch pollen allergen Bet v 1.0101. *PLoS One*. 2014;9(8):e104520.

12. Aalberse RC. Recombinant allergens need a reality check. *J Allergy Clin Immunol*. 2022;149(3):901-903.

13. Aalberse RC. Structural biology of allergens. *J Allergy Clin Immunol*. 2000;106(2):228-238.

14. Aalberse RC, Akkerdaas J, van Ree R. Cross-reactivity of IgE antibodies to allergens. *Allergy*. 2001;56(6):478-490.

15. Pascal M, Grishina G, Yang AC, et al. Molecular diagnosis of shrimp allergy: efficiency of several allergens to predict clinical reactivity. *J Allergy Clin Immunol Pract*. 2015;3(4):521-529.

16. Aalberse RC, Koshte V, Clemens JG. Immunoglobulin E antibodies that crossreact with vegetable foods, pollen, and Hymenoptera venom. *J Allergy Clin Immunol*. 1981;68(5):356-364.

17. Platts-Mills TA, Hilger C, Jappe U, et al. Carbohydrate epitopes currently recognized as targets for IgE antibodies. *Allergy*. 2021;76(8):2383-2394.

18. Platts-Mills TA, Schuyler AJ, Tripathi A, Commins SP. Anaphylaxis to the carbohydrate side chain alpha-gal. *Immunol Allergy Clin North Am*. 2015;35(2):247-260.

19. van Ree R. Carbohydrate epitopes and their relevance for the diagnosis and treatment of allergic diseases. *Int Arch Allergy Immunol*. 2002;129(3):189-197.

20. Platts-Mills TAE, Li RC, Keshavarz B, Smith AR, Wilson JM. Diagnosis and management of patients with the α-Gal syndrome. *J Allergy Clin Immunol Pract*. 2020;8(1):15-23.

21. Murangi T, Prakash P, Moreira BP, et al. Ascaris lumbricoides and ticks associated with sensitization to galactose α1,3-galactose and elicitation of the alpha-gal syndrome. *J Allergy Clin Immunol*. 2022;149(2):698-707.

22. Aalberse RC, Crameri R. IgE-binding epitopes: a reappraisal. *Allergy*. 2011;66(10):1261-1274.

23. Grüber C, Lau S, Dannemann A, Sommerfeld C, Wahn U, Aalberse RC. Down-regulation of IgE and IgG4 antibodies to tetanus toxin and diphtheria toxoid by covaccination with cellular Bordetella pertussis vaccine. *J Immunol*. 2001;167(4):2411-2417.

24. Caraballo L, Valenta R, Acevedo N, Zakzuk J. Are the terms major and minor allergens useful for precision allergology? *Front Immunol*. 2021;12:651500.

25. Breiteneder H, Mills EN. Molecular properties of food allergens. *J Allergy Clin Immunol*. 2005;115(1):14-23; quiz 24.

26. Virtanen T, Kinnunen T, Rytkönen-Nissinen M. Mammalian lipocalin allergens—insights into their enigmatic allergenicity. *Clin Exp Allergy*. 2012;42(4):494-504.

27. Westerhout J, Krone T, Snippe A, et al. Allergicenicity prediction of novel and modified proteins: not a mission impossible! Development of a Random Forest allergicenicity prediction model. *Regul Toxicol Pharmacol*. 2019;107:104422.

28. Posa D, Perna S, Resch Y, et al. Evolution and predictive value of IgE responses toward a comprehensive panel of house dust mite allergens during the first 2 decades of life. *J Allergy Clin Immunol*. 2017;139(2):541-549.

29. Sarzinszky E, Lupinek C, Vrtala S, et al. Expression in *Escherichia coli* and purification of folded rDer p 20, the arginine kinase from dermatophagoides pteronyssinus: a possible biomarker for allergic asthma. *Allergy Asthma Immunol Res*. 2021;13(1):154-163.

30. Siroux V, Boudier A, Nadif R, Lupinek C, Valenta R, Bousquet J. Association between asthma, rhinitis, and conjunctivitis morbidities with molecular IgE sensitization in adults. *Allergy*. 2019;74(4):824-827.

31. Nicolaou N, Poorafshar M, Murray C, et al. Allergy or tolerance in children sensitized to peanut: prevalence and differentiation using component-resolved diagnostics. *J Allergy Clin Immunol*. 2010;125(1):191-197.

32. Vieths S, Scheurer S, Ballmer-Weber B. Current understanding of cross-reactivity of food allergens and pollen. *Ann N Y Acad Sci*. 2002;964:47-68.

33. Sancho Al, Wangorsch A, Jensen BM, et al. Responsiveness of the major birch allergen Bet v 1 scaffold to the gastric environment: impact on structure and allergenic activity. *Mol Nutr Food Res*. 2011;55(11):1690-1699.

34. Reuter A, Lidholm J, Andersson K, et al. A critical assessment of allergen component-based in vitro diagnosis in cherry allergy across Europe. *Clin Exp Allergy*. 2006;36(6):815-823.
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