# C02 - PR-10-like allergens

## Heimo Breiteneder, Lorenz Aglas, Jörg Kleine-Tebbe, Christine Hafner

### Highlights

* The major birch pollen allergen Bet v 1 represents the phenotype of all PR-10-like allergens and is the primary sensitizer in birch pollen endemic regions.

* The presence of homologous allergens in Fagales tree pollen explains the IgE cross-reactivity between pollen from hazel, alder, beech, oak, hornbeam and chestnut.

* Minute amounts of PR-10-like allergens in raw fruits, nuts, vegetables and legumes can induce patient-individual patterns of oropharyngeal symptoms and sometimes severe allergic reactions in Bet v 1-sensitized individuals.

* Testing for Bet v 1-specific IgE is sufficient. The relevance of cross-reacting pollen or foods can be clinically clarified by seasonal and food-related symptoms without the need for further testing of Bet v 1 homologues.

## 1 – The PR-10 protein architecture

Bet v 1, the major allergen of birch pollen, was the first plant allergen and the first allergenic PR-10-like protein to be cloned and characterized (Table 72). The cDNA sequence coding for Bet v 1.0101 was discovered on July 3, 1988, and published in 1989, representing the most abundant isoform in birch pollen (50-70%).¹⁴²⁵

The protein architecture of Bet v 1 comprises a highly curved seven-stranded anti-parallel beta-sheet that embraces a 25 residue-long C-terminal alpha-helix.¹⁴²⁶ The beta-sheet and the C-terminal part of the long alpha-helix are separated by two consecutive alpha helices that connect the beta1- and beta2-strands. All these structural elements contribute to the formation of a large hydrophobic cavity. Structural information is available for various Bet v 1-homologous allergens from plant foods (Figure 146) and their overall similarity clearly illustrates the molecular basis for the cross-reactivity of these proteins. Although Bet v 1 contains a variety of different T cell epitopes, a major T cell epitope located at the C terminal amino acid residue positions 142-156 was recognized by T cells from 61% of birch pollen allergic individuals studied.¹⁴²⁷ This part of the molecule shares high sequence similarities with various Bet v 1-related tree pollen allergens. The extent of T cell cross-reactivity with Bet v 1-related food allergens also corresponded to the degree of sequence similarity of the food allergens' C-termini to the Bet v 1 amino acid residues 142-156.¹⁴²⁷ The sensitizing capacity of different Bet v 1 homologues from plant foods seems to correlate with the presence of immunodominant T cell epitopes.¹⁴²⁸ The Bet v 1-specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is variable and highly patient specific.¹⁴²⁹

<table><caption>TABLE 72 Protein characteristics</caption><thead><tr><th colspan="2">Protein characteristics</th></tr></thead><tbody><tr><td>Allergen source</td><td>Betula pendula (syn. B. verrucosa, B. alba), birch</td></tr><tr><td>Protein family</td><td>Bet v 1</td></tr><tr><td>Protein subfamily</td><td>PR-10</td></tr><tr><td>UniProt accession number</td><td>P15494</td></tr><tr><td>Crystal structure available</td><td>Yes</td></tr><tr><td>Molecular structure</td><td>Seven-stranded anti-parallel β-sheet flanked by three α-helices</td></tr><tr><td>Theoretical molecular weight</td><td>17439.63 Da</td></tr><tr><td>Molecular weight measured by MS</td><td>17439.6 +/- 0.3 Da</td></tr><tr><td>Length</td><td>159 amino acid residues</td></tr><tr><td>Ligand binding</td><td>Yes</td></tr><tr><td>Oligomerization state</td><td>Monomeric</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>0</td></tr><tr><td>Isoelectric point</td><td>5.39</td></tr><tr><td>Synthesis</td><td>In the cytoplasm of the vegetative cells of birch pollen</td></tr><tr><td>Distribution</td><td>Pollen is distributed into the environment by wind dispersal and can enter the indoor environment.</td></tr></tbody></table>

## 2 – The PR-10-like family of allergenic proteins

### 2.1. The Bet v 1-like superfamily

In 1980, pathogenesis-related proteins (PR proteins) were defined as "proteins encoded by the host plant but induced only in pathological or related situations" and subsequently grouped into families.¹⁴³⁰ Today, the list of PR proteins comprises 17 families.¹⁴³¹ When the sequence of Bet v 1 was discovered in 1989, the PR-10 family had not been defined yet, but it was noted that Bet v 1 was homologous to a PR protein from pea.¹⁴²⁵ Bet v 1 is constitutively expressed in pollen at rather high concentrations. Hence, the term PR-10 for the Bet v 1 homologous allergens is not entirely correct. These constitutively expressed proteins are referred to as PR-10-like proteins (Table 73). Since the discovery of Bet v 1, the number of sequences related to the Bet v 1 sequence has grown steadily and rapidly. The version 34.0 of the Pfam database attributes 114,208 sequences from 7,426 species to the Bet v 1-like superfamily (http://pfam.xfam.org/cluster/CL0209, accessed 12/2021) compared to 14,065 sequences from 1,452 species listed in the Pfam version 29.0 mentioned in the first edition of this book published in 2016. The member proteins of this superfamily are found in all three domains of life, i.e., archaea, bacteria and eukaryotes, and all share the Bet v 1 architecture.³⁴⁰ The Bet v 1-like superfamily of proteins comprises 25 families, one of which is the Bet v 1 family (http://pfam.xfam.org/family/PF00407). The Bet v 1 family in turn is composed of 11 subfamilies.³⁴⁰ One of these subfamilies is the PR-10 group of
# C02 - PR-10-like allergens

## Heimo Breiteneder, Lorenz Aglas, Jörg Kleine-Tebbe, Christine Hafner

### Highlights

* The major birch pollen allergen Bet v 1 represents the phenotype of all PR-10-like allergens and is the primary sensitizer in birch pollen endemic regions.

* The presence of homologous allergens in Fagales tree pollen explains the IgE cross-reactivity between pollen from hazel, alder, beech, oak, hornbeam and chestnut.

* Minute amounts of PR-10-like allergens in raw fruits, nuts, vegetables and legumes can induce patient-individual patterns of oropharyngeal symptoms and sometimes severe allergic reactions in Bet v 1-sensitized individuals.

* Testing for Bet v 1-specific IgE is sufficient. The relevance of cross-reacting pollen or foods can be clinically clarified by seasonal and food-related symptoms without the need for further testing of Bet v 1 homologues.

## 1 – The PR-10 protein architecture

Bet v 1, the major allergen of birch pollen, was the first plant allergen and the first allergenic PR-10-like protein to be cloned and characterized (Table 72). The cDNA sequence coding for Bet v 1.0101 was discovered on July 3, 1988, and published in 1989, representing the most abundant isoform in birch pollen (50-70%).¹⁴²⁵

The protein architecture of Bet v 1 comprises a highly curved seven-stranded anti-parallel beta-sheet that embraces a 25 residue-long C-terminal alpha-helix.¹⁴²⁶ The beta-sheet and the C-terminal part of the long alpha-helix are separated by two consecutive alpha helices that connect the beta1- and beta2-strands. All these structural elements contribute to the formation of a large hydrophobic cavity. Structural information is available for various Bet v 1-homologous allergens from plant foods (Figure 146) and their overall similarity clearly illustrates the molecular basis for the cross-reactivity of these proteins. Although Bet v 1 contains a variety of different T cell epitopes, a major T cell epitope located at the C terminal amino acid residue positions 142-156 was recognized by T cells from 61% of birch pollen allergic individuals studied.¹⁴²⁷ This part of the molecule shares high sequence similarities with various Bet v 1-related tree pollen allergens. The extent of T cell cross-reactivity with Bet v 1-related food allergens also corresponded to the degree of sequence similarity of the food allergens' C-termini to the Bet v 1 amino acid residues 142-156.¹⁴²⁷ The sensitizing capacity of different Bet v 1 homologues from plant foods seems to correlate with the presence of immunodominant T cell epitopes.¹⁴²⁸ The Bet v 1-specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is variable and highly patient specific.¹⁴²⁹

<table><caption>TABLE 72 Protein characteristics</caption><thead><tr><th colspan="2">Protein characteristics</th></tr></thead><tbody><tr><td>Allergen source</td><td>Betula pendula (syn. B. verrucosa, B. alba), birch</td></tr><tr><td>Protein family</td><td>Bet v 1</td></tr><tr><td>Protein subfamily</td><td>PR-10</td></tr><tr><td>UniProt accession number</td><td>P15494</td></tr><tr><td>Crystal structure available</td><td>Yes</td></tr><tr><td>Molecular structure</td><td>Seven-stranded anti-parallel β-sheet flanked by three α-helices</td></tr><tr><td>Theoretical molecular weight</td><td>17439.63 Da</td></tr><tr><td>Molecular weight measured by MS</td><td>17439.6 +/- 0.3 Da</td></tr><tr><td>Length</td><td>159 amino acid residues</td></tr><tr><td>Ligand binding</td><td>Yes</td></tr><tr><td>Oligomerization state</td><td>Monomeric</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>0</td></tr><tr><td>Isoelectric point</td><td>5.39</td></tr><tr><td>Synthesis</td><td>In the cytoplasm of the vegetative cells of birch pollen</td></tr><tr><td>Distribution</td><td>Pollen is distributed into the environment by wind dispersal and can enter the indoor environment.</td></tr></tbody></table>

## 2 – The PR-10-like family of allergenic proteins

### 2.1. The Bet v 1-like superfamily

In 1980, pathogenesis-related proteins (PR proteins) were defined as "proteins encoded by the host plant but induced only in pathological or related situations" and subsequently grouped into families.¹⁴³⁰ Today, the list of PR proteins comprises 17 families.¹⁴³¹ When the sequence of Bet v 1 was discovered in 1989, the PR-10 family had not been defined yet, but it was noted that Bet v 1 was homologous to a PR protein from pea.¹⁴²⁵ Bet v 1 is constitutively expressed in pollen at rather high concentrations. Hence, the term PR-10 for the Bet v 1 homologous allergens is not entirely correct. These constitutively expressed proteins are referred to as PR-10-like proteins (Table 73). Since the discovery of Bet v 1, the number of sequences related to the Bet v 1 sequence has grown steadily and rapidly. The version 34.0 of the Pfam database attributes 114,208 sequences from 7,426 species to the Bet v 1-like superfamily (http://pfam.xfam.org/cluster/CL0209, accessed 12/2021) compared to 14,065 sequences from 1,452 species listed in the Pfam version 29.0 mentioned in the first edition of this book published in 2016. The member proteins of this superfamily are found in all three domains of life, i.e., archaea, bacteria and eukaryotes, and all share the Bet v 1 architecture.³⁴⁰ The Bet v 1-like superfamily of proteins comprises 25 families, one of which is the Bet v 1 family (http://pfam.xfam.org/family/PF00407). The Bet v 1 family in turn is composed of 11 subfamilies.³⁴⁰ One of these subfamilies is the PR-10 group of
## PR-10 allergens of Fagales pollen

<figure><img src="image_258.png" /><figcaption>Bet v 1</figcaption></figure>

<figure><img src="image_259.png" /><figcaption>Fag s 1</figcaption></figure>

## Homologous PR-10 allergens found in plant foods

<figure><img src="image_260.png" /><figcaption>Fra a 1</figcaption></figure>

<figure><img src="image_261.png" /><figcaption>Mal d 1</figcaption></figure>

<figure><img src="image_262.png" /><figcaption>Pru av 1</figcaption></figure>

<figure><img src="image_263.png" /><figcaption>Pru p 1</figcaption></figure>

<figure><img src="image_264.png" /><figcaption>Act d 11</figcaption></figure>

<figure><img src="image_265.png" /><figcaption>Api g 1</figcaption></figure>

<figure><img src="image_266.png" /><figcaption>Ara h 8</figcaption></figure>

<figure><img src="image_267.png" /><figcaption>Gly m 4</figcaption></figure>

<figure><img src="image_268.png" /><figcaption>Vig r 6</figcaption></figure>

<figure><img src="image_269.png" /><figcaption>Cor a 1.04</figcaption></figure>

**FIGURE 146** Ribbon representations of birch pollen Bet v 1 (PDB 4A88) and homologues from beech pollen (Fag s 1; PDB 6ALK), strawberry (Fra a 1; PDB 6ST8), apple (Mal d 1; PDB 5MMU), cherry (Pru av 1; PDB 1E09), peach (Pru p 1; PDB 6Z98), green kiwifruit (Act d 11; PDB 4IGV), celery (Api g 1; PDB 2BK0), peanut (Ara h 8; PDB 4M9B), soybean (Gly m 4; PDB 2K7H), mung bean (Vig r 6; PDB 2FLH) and hazelnut (Cor a 1.04; PDB 6GQ9) rainbow-colored from blue at the N-terminus to red at the C-terminus. The 3D images were created with the molecular modeling system UCSF ChimeraX (https://www.rbvi.ucsf.edu/chimerax/).

proteins to which almost all of the Bet v 1 homologous allergens belong. There are only two examples of allergens that are members of other subfamilies, the kiwi allergen Act d 11³⁴² from the RRP/MLP (= ripening related proteins/major latex proteins) subfamily, and the mung bean allergen Vig r 6³⁴³ from the CSBP (= cytokinin-specific binding proteins) subfamily.

<caption>**TABLE 73** Characteristics of the PR-10-like family of allergenic proteins</caption>

* Common tertiary structure with a seven-stranded antiparallel β-sheet with a long C-terminal α-helix and two short α-helices
* Sequences with high identities
* Small cross-reactive molecules of around 17 kDa present in pollen of early flowering Fagales trees as well as in fruits, vegetables, nuts and seeds
* Binding of various ligands in a hydrophobic cavity

<table><caption>**TABLE 74** Bet v 1- homologous pollen allergens of Fagales pollen</caption><thead><tr><th>Botanical family</th><th>Botanical subfamily</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="5">Betulaceae</td><td rowspan="2">Betuloideae</td><td>Birch (Betula pendula)</td><td>Bet v 1</td></tr><tr><td>Alder (Alnus glutinosa)</td><td>Aln g 1</td></tr><tr><td rowspan="3">Coryloideae</td><td>Hazel (Corylus avellana)</td><td>Cor a 1</td></tr><tr><td>Hornbeam (Carpinus betulus)</td><td>Car b 1</td></tr><tr><td>Hop-hornbeam (Ostrya carpinifolia)</td><td>Ost c 1</td></tr><tr><td rowspan="5">Fagaceae</td><td rowspan="2">Fagoideae</td><td>Beech (Fagus silvatica)</td><td>Fag s 1</td></tr><tr><td>Oak (Quercus alba)</td><td>Que a 1</td></tr><tr><td rowspan="2">Quercoidae</td><td>Sawtooth oak (Quercus acutissima)</td><td>Que ac 1</td></tr><tr><td>Holly oak (Quercus ilex)</td><td>Que i 1</td></tr><tr><td rowspan="2">Castaneoideae</td><td rowspan="2">Chestnut (Castanea sativa)</td><td>Mongolian oak (Quercus mongolica)</td><td>Que m 1</td></tr><tr><td>Chestnut (Castanea sativa)</td><td>Cas s 1</td></tr></tbody></table>

## 2.2. Bet v 1-homologous allergens in Fagales tree pollen and plant foods

Birch pollen is one of the most common causes of IgE-mediated allergy in Northern and Central Europe as well as in North America. The major sensitizing allergen present in birch pollen is Bet v 1 to which 93% of individuals with birch pollen allergy produce specific IgE.¹⁴³² Birch belongs to the botanical order Fagales, which comprises seven families. Allergies have been strongly associated with pollen produced by the early flowering trees of the families Betulaceae and the Fagaceae (Table 74 and Figure 147). In general, allergic reactions to Fagales pollen are initiated by independent sensitization to pollen of members of the Betuloideae or Coryloideae subfamilies. However, 25% of the IgE epitopes of the Betuloideae and the Coryloideae pollen allergens are unique for the respective subfamily, whereas pollen allergens from the Fagaceae are generally cross-reactive.¹⁴³³

Homologues of Bet v 1 have also been identified in a wide range of plant foods.¹⁴³⁴ The most frequently observed clinical entity is caused by IgE antibodies that cross-react between Bet v 1 and its homologues in fruits, nuts, seeds and vegetables. They induce predominantly oropharyngeal symptoms, which are summarized by the term oral allergy syndrome (OAS)³² Severe reactions to Gly m 4, the Bet v 1 homologue from soybean (see chapter B17), have been observed in a subpopulation of Bet v 1-allergic individuals.¹²¹⁷ Bet v 1-allergic patients are at risk to acquire various plant food allergies, and even to react to novel foods without prior exposure.³² In contrast to Bet v 1, Bet v 1-related food allergens are regarded as incapable of sensitizing
## PR-10 allergens of Fagales pollen

<figure><img src="image_264.png" /><figcaption>Bet v 1</figcaption></figure>

<figure><img src="image_265.png" /><figcaption>Fag s 1</figcaption></figure>

## Homologous PR-10 allergens found in plant foods

<figure><img src="image_266.png" /><figcaption>Fra a 1</figcaption></figure>

<figure><img src="image_267.png" /><figcaption>Mal d 1</figcaption></figure>

<figure><img src="image_268.png" /><figcaption>Pru av 1</figcaption></figure>

<figure><img src="image_269.png" /><figcaption>Pru p 1</figcaption></figure>

<figure><img src="image_270.png" /><figcaption>Act d 11</figcaption></figure>

<figure><img src="image_271.png" /><figcaption>Api g 1</figcaption></figure>

<figure><img src="image_272.png" /><figcaption>Ara h 8</figcaption></figure>

<figure><img src="image_273.png" /><figcaption>Gly m 4</figcaption></figure>

<figure><img src="image_274.png" /><figcaption>Vig r 6</figcaption></figure>

<figure><img src="image_275.png" /><figcaption>Cor a 1.04</figcaption></figure>

**FIGURE 146** Ribbon representations of birch pollen Bet v 1 (PDB 4A88) and homologues from beech pollen (Fag s 1; PDB 6ALK), strawberry (Fra a 1; PDB 6ST8), apple (Mal d 1; PDB 5MMU), cherry (Pru av 1; PDB 1E09), peach (Pru p 1; PDB 6Z98), green kiwifruit (Act d 11; PDB 4IGV), celery (Api g 1; PDB 2BK0), peanut (Ara h 8; PDB 4M9B), soybean (Gly m 4; PDB 2K7H), mung bean (Vig r 6; PDB 2FLH) and hazelnut (Cor a 1.04; PDB 6GQ9) rainbow-colored from blue at the N-terminus to red at the C-terminus. The 3D images were created with the molecular modeling system UCSF ChimeraX (https://www.rbvi.ucsf.edu/chimerax/).

proteins to which almost all of the Bet v 1 homologous allergens belong. There are only two examples of allergens that are members of other subfamilies, the kiwi allergen Act d 11³⁴² from the RRP/MLP (= ripening related proteins/major latex proteins) subfamily, and the mung bean allergen Vig r 6³⁴³ from the CSBP (= cytokinin-specific binding proteins) subfamily.

<caption>**TABLE 73** Characteristics of the PR-10-like family of allergenic proteins</caption>

* Common tertiary structure with a seven-stranded antiparallel β-sheet with a long C-terminal α-helix and two short α-helices
* Sequences with high identities
* Small cross-reactive molecules of around 17 kDa present in pollen of early flowering Fagales trees as well as in fruits, vegetables, nuts and seeds
* Binding of various ligands in a hydrophobic cavity

<table><caption>**TABLE 74** Bet v 1- homologous pollen allergens of Fagales pollen</caption><thead><tr><th>Botanical family</th><th>Botanical subfamily</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="5">Betulaceae</td><td rowspan="2">Betuloideae</td><td>Birch (Betula pendula)</td><td>Bet v 1</td></tr><tr><td>Alder (Alnus glutinosa)</td><td>Aln g 1</td></tr><tr><td rowspan="3">Coryloideae</td><td>Hazel (Corylus avellana)</td><td>Cor a 1</td></tr><tr><td>Hornbeam (Carpinus betulus)</td><td>Car b 1</td></tr><tr><td>Hop-hornbeam (Ostrya carpinifolia)</td><td>Ost c 1</td></tr><tr><td rowspan="5">Fagaceae</td><td rowspan="2">Fagoideae</td><td>Beech (Fagus silvatica)</td><td>Fag s 1</td></tr><tr><td>Oak (Quercus alba)</td><td>Que a 1</td></tr><tr><td rowspan="2">Quercoidae</td><td>Sawtooth oak (Quercus acutissima)</td><td>Que ac 1</td></tr><tr><td>Holly oak (Quercus ilex)</td><td>Que i 1</td></tr><tr><td rowspan="2">Castaneoideae</td><td rowspan="2">Chestnut (Castanea sativa)</td><td>Mongolian oak (Quercus mongolica)</td><td>Que m 1</td></tr><tr><td>Chestnut (Castanea sativa)</td><td>Cas s 1</td></tr></tbody></table>

## 2.2. Bet v 1-homologous allergens in Fagales tree pollen and plant foods

Birch pollen is one of the most common causes of IgE-mediated allergy in Northern and Central Europe as well as in North America. The major sensitizing allergen present in birch pollen is Bet v 1 to which 93% of individuals with birch pollen allergy produce specific IgE.¹⁴³² Birch belongs to the botanical order Fagales, which comprises seven families. Allergies have been strongly associated with pollen produced by the early flowering trees of the families Betulaceae and the Fagaceae (Table 74 and Figure 147). In general, allergic reactions to Fagales pollen are initiated by independent sensitization to pollen of members of the Betuloideae or Coryloideae subfamilies. However, 25% of the IgE epitopes of the Betuloideae and the Coryloideae pollen allergens are unique for the respective subfamily, whereas pollen allergens from the Fagaceae are generally cross-reactive.¹⁴³³

Homologues of Bet v 1 have also been identified in a wide range of plant foods.¹⁴³⁴ The most frequently observed clinical entity is caused by IgE antibodies that cross-react between Bet v 1 and its homologues in fruits, nuts, seeds and vegetables. They induce predominantly oropharyngeal symptoms, which are summarized by the term oral allergy syndrome (OAS)³² Severe reactions to Gly m 4, the Bet v 1 homologue from soybean (see chapter B17), have been observed in a subpopulation of Bet v 1-allergic individuals.¹²¹⁷ Bet v 1-allergic patients are at risk to acquire various plant food allergies, and even to react to novel foods without prior exposure.³² In contrast to Bet v 1, Bet v 1-related food allergens are regarded as incapable of sensitizing
<figure><img src="image_270.png" /><figcaption>FIGURE 147 Tree pollen sources of Bet v 1 homologous PR-10-like allergens. Official allergen names assigned by the WHO/IUIS Allergen Nomenclature Sub-Committee (allergen.org) are shown in Table 74. Extensive IgE cross-reactivity between the various pollen PR-10-like allergens has been determined.</figcaption></figure>

predisposed individuals. The few exceptions that were described include Dau c 1 from carrot,¹⁴³⁵⁻¹⁴³⁷ and Cor a 1 from hazelnut.¹⁴³⁸

The list of plant food allergens to which Bet v 1-allergic individuals may react is quite varied and most likely connected to the variation in individual IgE epitope patterns described.¹⁴²⁹ Interestingly, most of the Bet v 1 cross-reactive allergens are found in fruits of the *Rosaceae*, in vegetables of the *Apiaceae*, and in seeds of the *Fabaceae* family (Table 75 and Figure 148).

Amino acid sequence identities between PR-10-like pollen allergens are between 49 and 96% (Table 76) and identities between Bet v 1 and plant food allergens fall between 17 and 68% (Table 77). There is also a range of plant foods that contain cross-reactive Bet v 1 homologues that have not yet received an official allergen nomenclature designation including almond, asparagus, parsley, ginseng, plum, nectarine, fig, mango, persimmon, jackfruit, and chickpea.

## 2.3. Ligand binding of PR-10-like proteins

The large hydrophobic cavity at the core of most PR-10-like allergens enables the binding of several physiological and experimental ligands, mainly low molecular weight compounds of the chemical classes of cytokinins, flavonoids and sterols.³⁴⁰,¹⁴³⁹ Ligand binding was described for Fagales pollen allergens from birch, hazel, and beech but also for Bet v 1-homologous allergens from peanut, strawberry, cherry and peach. Although ligand binding in PR-10-like allergens appears to occur in a promiscuous, nonspecific way, the ligand preference varies greatly among the different proteins and among isoforms, as indicated by differences in binding affinities.⁴⁷⁶,¹⁴⁴⁰,¹⁴⁴¹ The role of ligand binding of PR-10-like allergens ranges from an involvement in flavonoid biosynthesis (polyphenolic plant metabolites involved in color/flavor production and UV protection) to more generalized mechanisms in plant development, defense and reproduction.¹⁴⁴²⁻¹⁴⁴⁴ A detailed description of ligand classes interacting with PR-10 allergens is provided in (Table 78).

In most PR-10-like allergens, ligand-binding results in a stabilization of the protein via structure rigidification, which, in case of Bet v 1, leads to increased thermal stability and a decreased proteolytic susceptibility without changing its secondary structure. However, clinical practice shows us that the majority of Bet v-1-homologues containing foods will only lead to symptoms when consumed raw

<table><caption>TABLE 75 Bet v 1- homologous allergens of plants</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="7">Rosaceae</td><td>Strawberry (Fragaria x ananassa)</td><td>Fra a 1</td></tr><tr><td>Apple (Malus domestica)</td><td>Mal d 1</td></tr><tr><td>Apricot (Prunus armeniaca)</td><td>Pru ar 1</td></tr><tr><td>Cherry (Prunus avium)</td><td>Pru av 1</td></tr><tr><td>Peach (Prunus persica)</td><td>Pru p 1</td></tr><tr><td>Pear (Pyrus communis)</td><td>Pyr c 1</td></tr><tr><td>Raspberry (Rubus idaeus)</td><td>Rub i 1</td></tr><tr><td rowspan="2">Actinidiaceae</td><td>Golden kiwifruit (Actinidia chinensis)</td><td>Act c 8</td></tr><tr><td>Green kiwifruit (Actinidia deliciosa)</td><td>Act d 8</td></tr><tr><td rowspan="2">Apiaceae</td><td>Celery (Apium graveolens)</td><td>Act d 11</td></tr><tr><td>Carrot (Daucus carota)</td><td>Api g 1<br>Dau c 1</td></tr><tr><td rowspan="4">Fabaceae</td><td>Peanut (Arachis hypogaea)</td><td>Ara h 8</td></tr><tr><td>Soybean (Glycine max)</td><td>Gly m 4</td></tr><tr><td>Mung bean (Vigna radiata)</td><td>Vig r 1<br>Vig r 6</td></tr><tr><td>Hazelnut (Corylus avellana)</td><td>Cor a 1.04</td></tr><tr><td>Corylaceae</td><td>Tomato (Solanum lycopersicum)</td><td>Sola 1 4</td></tr><tr><td>Juglandaceae</td><td>English walnut (Juglans regia)</td><td>Jug r 5</td></tr><tr><td>Cannabaceae</td><td>Indian hemp (Cannabis sativa)</td><td>Can s 5</td></tr></tbody></table>

(see also chapter 3.3). Two naturally occurring, physiological ligands were described for Bet v 1, the glycosylated flavonoid quercetin-3-O-sophoroside and phytoprostanes, which are pollen-associated lipid mediators.⁴⁷¹,¹⁴⁴⁵ The stabilization of proteolytic cleavage sites by ligand binding affects the presentation of Bet v 1 peptides to T cells via the major histocompatibility complex class II molecules. The phytoprostane PPE1 hereby fulfills a dual role, on the one hand, by stabilising Bet v 1 against proteolytic degradation and, on the other hand, by modulating the activity of proteolytic enzymes. The increased proteolytic resistance mediated by these ligands and the altered presentation of T cell peptides suggests a contribution of ligands to allergic sensitization.
<figure><img src="image_276.png" /><figcaption>FIGURE 147 Tree pollen sources of Bet v 1 homologous PR-10-like allergens. Official allergen names assigned by the WHO/IUIS Allergen Nomenclature Sub-Committee (allergen.org) are shown in Table 74. Extensive IgE cross-reactivity between the various pollen PR-10-like allergens has been determined.</figcaption></figure>

predisposed individuals. The few exceptions that were described include Dau c 1 from carrot,¹⁴³⁵⁻¹⁴³⁷ and Cor a 1 from hazelnut.¹⁴³⁸

The list of plant food allergens to which Bet v 1-allergic individuals may react is quite varied and most likely connected to the variation in individual IgE epitope patterns described.¹⁴²⁹ Interestingly, most of the Bet v 1 cross-reactive allergens are found in fruits of the *Rosaceae*, in vegetables of the *Apiaceae*, and in seeds of the *Fabaceae* family (Table 75 and Figure 148).

Amino acid sequence identities between PR-10-like pollen allergens are between 49 and 96% (Table 76) and identities between Bet v 1 and plant food allergens fall between 17 and 68% (Table 77). There is also a range of plant foods that contain cross-reactive Bet v 1 homologues that have not yet received an official allergen nomenclature designation including almond, asparagus, parsley, ginseng, plum, nectarine, fig, mango, persimmon, jackfruit, and chickpea.

## 2.3. Ligand binding of PR-10-like proteins

The large hydrophobic cavity at the core of most PR-10-like allergens enables the binding of several physiological and experimental ligands, mainly low molecular weight compounds of the chemical classes of cytokinins, flavonoids and sterols.³⁴⁰,¹⁴³⁹ Ligand binding was described for Fagales pollen allergens from birch, hazel, and beech but also for Bet v 1-homologous allergens from peanut, strawberry, cherry and peach. Although ligand binding in PR-10-like allergens appears to occur in a promiscuous, nonspecific way, the ligand preference varies greatly among the different proteins and among isoforms, as indicated by differences in binding affinities.⁴⁷⁶,¹⁴⁴⁰,¹⁴⁴¹ The role of ligand binding of PR-10-like allergens ranges from an involvement in flavonoid biosynthesis (polyphenolic plant metabolites involved in color/flavor production and UV protection) to more generalized mechanisms in plant development, defense and reproduction.¹⁴⁴²⁻¹⁴⁴⁴ A detailed description of ligand classes interacting with PR-10 allergens is provided in (Table 78).

In most PR-10-like allergens, ligand-binding results in a stabilization of the protein via structure rigidification, which, in case of Bet v 1, leads to increased thermal stability and a decreased proteolytic susceptibility without changing its secondary structure. However, clinical practice shows us that the majority of Bet v-1-homologues containing foods will only lead to symptoms when consumed raw

<table><caption>TABLE 75 Bet v 1- homologous allergens of plants</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td rowspan="7">Rosaceae</td><td>Strawberry (Fragaria x ananassa)</td><td>Fra a 1</td></tr><tr><td>Apple (Malus domestica)</td><td>Mal d 1</td></tr><tr><td>Apricot (Prunus armeniaca)</td><td>Pru ar 1</td></tr><tr><td>Cherry (Prunus avium)</td><td>Pru av 1</td></tr><tr><td>Peach (Prunus persica)</td><td>Pru p 1</td></tr><tr><td>Pear (Pyrus communis)</td><td>Pyr c 1</td></tr><tr><td>Raspberry (Rubus idaeus)</td><td>Rub i 1</td></tr><tr><td rowspan="2">Actinidiaceae</td><td>Golden kiwifruit (Actinidia chinensis)</td><td>Act c 8</td></tr><tr><td>Green kiwifruit (Actinidia deliciosa)</td><td>Act d 8</td></tr><tr><td rowspan="2">Apiaceae</td><td>Celery (Apium graveolens)</td><td>Act d 11</td></tr><tr><td>Carrot (Daucus carota)</td><td>Api g 1<br>Dau c 1</td></tr><tr><td rowspan="4">Fabaceae</td><td>Peanut (Arachis hypogaea)</td><td>Ara h 8</td></tr><tr><td>Soybean (Glycine max)</td><td>Gly m 4</td></tr><tr><td>Mung bean (Vigna radiata)</td><td>Vig r 1<br>Vig r 6</td></tr><tr><td>Hazelnut (Corylus avellana)</td><td>Cor a 1.04</td></tr><tr><td>Corylaceae</td><td>Tomato (Solanum lycopersicum)</td><td>Sola 1 4</td></tr><tr><td>Juglandaceae</td><td>English walnut (Juglans regia)</td><td>Jug r 5</td></tr><tr><td>Cannabaceae</td><td>Indian hemp (Cannabis sativa)</td><td>Can s 5</td></tr></tbody></table>

(see also chapter 3.3). Two naturally occurring, physiological ligands were described for Bet v 1, the glycosylated flavonoid quercetin-3-O-sophoroside and phytoprostanes, which are pollen-associated lipid mediators.⁴⁷¹,¹⁴⁴⁵ The stabilization of proteolytic cleavage sites by ligand binding affects the presentation of Bet v 1 peptides to T cells via the major histocompatibility complex class II molecules. The phytoprostane PPE1 hereby fulfills a dual role, on the one hand, by stabilising Bet v 1 against proteolytic degradation and, on the other hand, by modulating the activity of proteolytic enzymes. The increased proteolytic resistance mediated by these ligands and the altered presentation of T cell peptides suggests a contribution of ligands to allergic sensitization.
<figure><img src="image_271.png" /><figcaption>FIGURE 148 Plant food sources of PR-10-like allergens. Official allergen names are shown in Table 75. These PR-10-like allergens for which IgE cross-reactivity with the major birch pollen allergen Bet v 1 has been determined are found in the botanical families Rosaceae (apple, pear, cherry, apricot, peach, strawberry, raspberry), Actinidiaceae (golden kiwifruit, green kiwifruit), Apiaceae (celeriac, carrot), Fabaceae (peanut, soybean, mung bean), Solanaceae (tomato), Corylaceae (hazelnut), and Juglandaceae (walnut). Inhibition experiments have also indicated the presence of allergenic members of the PR-10 family in plum, nectarine, fig, mango, persimmon, jackfruit, chickpea, potato, chicory, fennel, poppy seeds, chamomile, parsley, anise seeds, cumin seeds, and coriander seeds (not shown).</figcaption></figure>

<table><caption>TABLE 76 Amino acid sequence identities (%) between PR-10 allergens of Fagales pollen</caption><thead><tr><th></th><th>Aln g 1</th><th>Bet v 1</th><th>Car b 1</th><th>Cas s 1</th><th>Cor a 1</th><th>Fag s 1</th><th>Ost c 1</th><th>Que a 1</th><th>Que ac 1</th><th>Que i 1</th><th>Que u 1</th></tr></thead><tbody><tr><td>Aln g 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bet v 1</td><td>81</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Car b 1</td><td>74</td><td>73</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cas s 1</td><td>59</td><td>59</td><td>53</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cor a 1</td><td>76</td><td>72</td><td>88</td><td>58</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Fag s 1</td><td>64</td><td>66</td><td>58</td><td>67</td><td>62</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ost c 1</td><td>76</td><td>74</td><td>96</td><td>54</td><td>91</td><td>60</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Que a 1</td><td>60</td><td>58</td><td>52</td><td>89</td><td>55</td><td>67</td><td>53</td><td></td><td></td><td></td><td></td></tr><tr><td>Que ac 1</td><td>59</td><td>58</td><td>49</td><td>85</td><td>53</td><td>63</td><td>50</td><td>83</td><td></td><td></td><td></td></tr><tr><td>Que i 1</td><td>61</td><td>59</td><td>51</td><td>90</td><td>55</td><td>67</td><td>53</td><td>89</td><td>84</td><td></td><td></td></tr><tr><td>Que m 1</td><td>62</td><td>58</td><td>56</td><td>76</td><td>59</td><td>67</td><td>57</td><td>75</td><td>73</td><td>74</td><td></td></tr></tbody><tfoot><tr><td colspan="12">Percent identity matrix was created using Clustal2.1 (EMBL-EBI)</td></tr><tr><td colspan="12">50 95</td></tr></tfoot></table>

## 3 - Clinical relevance

### 3.1. General importance

Bet v 1 is regarded as a marker allergen for a primary sensitization to pollen of birch and other Fagales trees (e.g., alder, hazel, hornbeam, beech, oak), and an indicator of cross-reactivity to a number of related major pollen and plant food allergens. Clinically, Bet v 1 and its homologues in pollen represent important inhalant allergens, and are considered important inducers of birch pollen-associated plant food allergies.

### 3.2. Epidemiology

According to the European Respiratory Health Survey published in 2007, the prevalence of sensitization to birch pollen was on average 6.4% with a maximum of 22.4% in Northern Europe.⁶²⁶ Sensitization was generally high in Northern and Central Europe and low in the South of Europe. A large nationwide study in Germany on a representative sample of children and adolescents published in 2013 revealed the presence of IgE specific for birch pollen allergens in 15% of the individuals in the age group of 3 to 17 years.⁷⁹⁸ In the age group of 13 to 17 years, 15.7% of the girls and 21.7% of
<figure><img src="image_277.png" /><figcaption>FIGURE 148 Plant food sources of PR-10-like allergens. Official allergen names are shown in Table 75. These PR-10-like allergens for which IgE cross-reactivity with the major birch pollen allergen Bet v 1 has been determined are found in the botanical families Rosaceae (apple, pear, cherry, apricot, peach, strawberry, raspberry), Actinidiaceae (golden kiwifruit, green kiwifruit), Apiaceae (celeriac, carrot), Fabaceae (peanut, soybean, mung bean), Solanaceae (tomato), Corylaceae (hazelnut), and Juglandaceae (walnut). Inhibition experiments have also indicated the presence of allergenic members of the PR-10 family in plum, nectarine, fig, mango, persimmon, jackfruit, chickpea, potato, chicory, fennel, poppy seeds, chamomile, parsley, anise seeds, cumin seeds, and coriander seeds (not shown).</figcaption></figure>

<table><caption>TABLE 76 Amino acid sequence identities (%) between PR-10 allergens of Fagales pollen</caption><thead><tr><th></th><th>Aln g 1</th><th>Bet v 1</th><th>Car b 1</th><th>Cas s 1</th><th>Cor a 1</th><th>Fag s 1</th><th>Ost c 1</th><th>Que a 1</th><th>Que ac 1</th><th>Que i 1</th><th>Que u 1</th></tr></thead><tbody><tr><td>Aln g 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bet v 1</td><td>81</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Car b 1</td><td>74</td><td>73</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cas s 1</td><td>59</td><td>59</td><td>53</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cor a 1</td><td>76</td><td>72</td><td>88</td><td>58</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Fag s 1</td><td>64</td><td>66</td><td>58</td><td>67</td><td>62</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ost c 1</td><td>76</td><td>74</td><td>96</td><td>54</td><td>91</td><td>60</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Que a 1</td><td>60</td><td>58</td><td>52</td><td>89</td><td>55</td><td>67</td><td>53</td><td></td><td></td><td></td><td></td></tr><tr><td>Que ac 1</td><td>59</td><td>58</td><td>49</td><td>85</td><td>53</td><td>63</td><td>50</td><td>83</td><td></td><td></td><td></td></tr><tr><td>Que i 1</td><td>61</td><td>59</td><td>51</td><td>90</td><td>55</td><td>67</td><td>53</td><td>89</td><td>84</td><td></td><td></td></tr><tr><td>Que m 1</td><td>62</td><td>58</td><td>56</td><td>76</td><td>59</td><td>67</td><td>57</td><td>75</td><td>73</td><td>74</td><td></td></tr></tbody><tfoot><tr><td colspan="12">Percent identity matrix was created using Clustal2.1 (EMBL-EBI)</td></tr><tr><td colspan="12">50 95</td></tr></tfoot></table>

## 3 – Clinical relevance

### 3.1. General importance

Bet v 1 is regarded as a marker allergen for a primary sensitization to pollen of birch and other Fagales trees (e.g., alder, hazel, hornbeam, beech, oak), and an indicator of cross-reactivity to a number of related major pollen and plant food allergens. Clinically, Bet v 1 and its homologues in pollen represent important inhalant allergens, and are considered important inducers of birch pollen-associated plant food allergies.

### 3.2. Epidemiology

According to the European Respiratory Health Survey published in 2007, the prevalence of sensitization to birch pollen was on average 6.4% with a maximum of 22.4% in Northern Europe.⁶²⁶ Sensitization was generally high in Northern and Central Europe and low in the South of Europe. A large nationwide study in Germany on a representative sample of children and adolescents published in 2013 revealed the presence of IgE specific for birch pollen allergens in 15% of the individuals in the age group of 3 to 17 years.⁷⁹⁸ In the age group of 13 to 17 years, 15.7% of the girls and 21.7% of
<table><caption>TABLE 77 Amino acid sequence identities (%) between Bet v 1 and homologous PR-10 allergens found in plants</caption><thead><tr><th></th><th>Act c 8</th><th>Act d 8</th><th>Act d 11</th><th>Api g 1</th><th>Ara h 8</th><th>Bet v 1</th><th>Cans 5</th><th>Cor a 1.04</th><th>Dauc s 1</th><th>Fra a 1</th><th>Gly m 4</th><th>Jug r 5</th><th>Mal d 1</th><th>Pru ar 1</th><th>Pru av 1</th><th>Pru p 1</th><th>Pyr c 1</th><th>Rub i 1</th><th>Sola l 4</th><th>Vi g r 1</th><th>Vi g r 6</th></tr></thead><tbody><tr><td>Act c 8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Act d 8</td><td>70</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Act d 11</td><td>21</td><td>21</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Api g 1</td><td>45</td><td>48</td><td>13</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ara h 8</td><td>45</td><td>39</td><td>18</td><td>38</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bet v 1</td><td>50</td><td>50</td><td>17</td><td>42</td><td>47</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cans 5</td><td>50</td><td>52</td><td>20</td><td>42</td><td>44</td><td>54</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cor a 1.04</td><td>53</td><td>54</td><td>19</td><td>39</td><td>54</td><td>68</td><td>58</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Dauc s 1</td><td>45</td><td>47</td><td>16</td><td>81</td><td>36</td><td>38</td><td>38</td><td>37</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Fra a 1</td><td>53</td><td>54</td><td>16</td><td>44</td><td>51</td><td>54</td><td>64</td><td>61</td><td>41</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Gly m 4</td><td>47</td><td>45</td><td>18</td><td>39</td><td>71</td><td>47</td><td>50</td><td>58</td><td>37</td><td>52</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Jug r 5</td><td>57</td><td>55</td><td>19</td><td>42</td><td>54</td><td>67</td><td>61</td><td>68</td><td>40</td><td>69</td><td>54</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mal d 1</td><td>53</td><td>50</td><td>18</td><td>39</td><td>50</td><td>56</td><td>61</td><td>62</td><td>37</td><td>77</td><td>53</td><td>74</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pru ar 1</td><td>64</td><td>61</td><td>18</td><td>46</td><td>50</td><td>60</td><td>59</td><td>64</td><td>44</td><td>73</td><td>51</td><td>73</td><td>72</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pru av 1</td><td>56</td><td>54</td><td>18</td><td>42</td><td>51</td><td>59</td><td>64</td><td>66</td><td>39</td><td>81</td><td>53</td><td>74</td><td>85</td><td>76</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pru p 1</td><td>57</td><td>55</td><td>18</td><td>42</td><td>50</td><td>59</td><td>65</td><td>64</td><td>40</td><td>83</td><td>53</td><td>74</td><td>83</td><td>77</td><td>98</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pyr c 1</td><td>52</td><td>50</td><td>20</td><td>39</td><td>50</td><td>57</td><td>60</td><td>62</td><td>38</td><td>76</td><td>52</td><td>75</td><td>94</td><td>73</td><td>84</td><td>84</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Rub i 1</td><td>52</td><td>57</td><td>15</td><td>43</td><td>47</td><td>55</td><td>59</td><td>62</td><td>40</td><td>69</td><td>48</td><td>71</td><td>68</td><td>72</td><td>72</td><td>72</td><td>68</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sola l 4</td><td>48</td><td>48</td><td>19</td><td>39</td><td>41</td><td>44</td><td>49</td><td>50</td><td>35</td><td>49</td><td>43</td><td>51</td><td>44</td><td>51</td><td>49</td><td>50</td><td>43</td><td>47</td><td></td><td></td><td></td><td></td></tr><tr><td>Vig r 1</td><td>43</td><td>44</td><td>17</td><td>39</td><td>67</td><td>44</td><td>46</td><td>54</td><td>35</td><td>46</td><td>76</td><td>52</td><td>47</td><td>48</td><td>47</td><td>47</td><td>45</td><td>46</td><td>41</td><td></td><td></td><td></td></tr><tr><td>Vig r 6</td><td>23</td><td>23</td><td>15</td><td>20</td><td>20</td><td>26</td><td>25</td><td>19</td><td>18</td><td>24</td><td>24</td><td>25</td><td>23</td><td>25</td><td>22</td><td>23</td><td>22</td><td>22</td><td>24</td><td>23</td><td></td><td></td></tr></tbody></table>

Percent identity matrix was created using Clustal2.1 (EMBL-EBI)

<table><caption>TABLE 78 Reported ligand classes interacting with PR-10 allergens</caption><thead><tr><th>Ligand chemical class</th><th>Reported allergens</th></tr></thead><tbody><tr><td>Animal steroid hormones</td><td>Ara h 8, Cor a 1, Que a 1</td></tr><tr><td>Brassinosteroid (analog)</td><td>Bet v 1, Cor a 1, Fag s 1, Pru av 1, Que a 1</td></tr><tr><td>Cytokinin</td><td>Cor a 1, Bet v 1, Fag s 1, Pru p 1</td></tr><tr><td>Extrinsic and non-physiological ligands</td><td>Bet v 1, Fag s 1</td></tr><tr><td>Fatty acids</td><td>Bet v 1, Cor a 1, Que a 1</td></tr><tr><td>Flavonoids</td><td>Ara h 8, Bet v 1, Cor a 1, Fag s 1, Fra a 1, Que a 1</td></tr><tr><td>Organic compounds</td><td>Ara h 8, Cor a 1, Fag s 1</td></tr><tr><td>Phytoprostanes</td><td>Bet v 1</td></tr></tbody></table>

the boys had IgE specific for birch pollen allergens. IgE sensitization to birch pollen allergens in adults (age 19 to 79 years) was found to be 17.4% and sensitization to Bet v 1 15.2%.1226 Approximately half of all sensitized individuals will develop symptoms such as allergic rhinoconjunctivitis or allergic asthma.798

According to the National Health and Nutrition Examination Survey (NHANES) 2005-2006 sensitization to birch pollen in the United States was on average 10% with slightly higher sensitization rates of 12.3% and 11.8% in the Northeast and Western regions, respectively.627 The NHANES 2005-2006 data also demonstrated racial/ethnic differences. Sensitization to birch pollen was significantly

higher in non-Hispanic blacks (14%) compared with non-Hispanic whites (9.1%).627

Oak, hazel and alder pollen account for sensitization to Bet v 1-homologous proteins in East Asia, with oak tree pollen dominating over pollen of other tree species due to climate change1446. The IgE-sensitization rate to oak pollen has more than doubled from 4.7% in 1998 to 9.8% in 2019 in children living in the Seoul metropolitan area.1446

### 3.3. Symptoms (respiratory allergy)

Typical mucosal symptoms of tree pollen allergy occur during springtime in the respective regions (in Central Europe between February and May) with maxima depending on the current climate and the region's altitude:

* itchiness, redness, tearing of the eyes

* itch in the nose, oropharyngeal itch, (repeated) sneezing, runny and/or stuffy nose

* occasionally dry cough (particularly during or shortly after exercise), dyspnea, pressure sensation on the chest, wheezing, bronchial secretion and difficulty in breathing as indicators for increasingly affected lower airways (asthmatic lower airway inflammation)

* occasionally flu-like symptoms such as fatigue, body aches and headaches

Clinical diagnoses of intermittent (seasonal) rhinoconjunctivitis and allergic asthma due to birch pollen become more likely when mucosal symptoms occur during the same season in subsequent
<table><caption>TABLE 77 Amino acid sequence identities (%) between Bet v 1 and homologous PR-10 allergens found in plants</caption><thead><tr><th></th><th>Act c 8</th><th>Act d 8</th><th>Act d 11</th><th>Api g 1</th><th>Ara h 8</th><th>Bet v 1</th><th>Cans 5</th><th>Cor a 1.04</th><th>Dauc s 1</th><th>Fra a 1</th><th>Gly m 4</th><th>Jug r 5</th><th>Mal d 1</th><th>Pru ar 1</th><th>Pru av 1</th><th>Pru p 1</th><th>Pyr c 1</th><th>Rub i 1</th><th>Sola l 4</th><th>Vi g r 1</th><th>Vi g r 6</th></tr></thead><tbody><tr><td>Act c 8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Act d 8</td><td>70</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Act d 11</td><td>21</td><td>21</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Api g 1</td><td>45</td><td>48</td><td>13</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Ara h 8</td><td>45</td><td>39</td><td>18</td><td>38</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bet v 1</td><td>50</td><td>50</td><td>17</td><td>42</td><td>47</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cans 5</td><td>50</td><td>52</td><td>20</td><td>42</td><td>44</td><td>54</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Cor a 1.04</td><td>53</td><td>54</td><td>19</td><td>39</td><td>54</td><td>68</td><td>58</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Dauc s 1</td><td>45</td><td>47</td><td>16</td><td>81</td><td>36</td><td>38</td><td>38</td><td>37</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Fra a 1</td><td>53</td><td>54</td><td>16</td><td>44</td><td>51</td><td>54</td><td>64</td><td>61</td><td>41</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Gly m 4</td><td>47</td><td>45</td><td>18</td><td>39</td><td>71</td><td>47</td><td>50</td><td>58</td><td>37</td><td>52</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Jug r 5</td><td>57</td><td>55</td><td>19</td><td>42</td><td>54</td><td>67</td><td>61</td><td>68</td><td>40</td><td>69</td><td>54</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mal d 1</td><td>53</td><td>50</td><td>18</td><td>39</td><td>50</td><td>56</td><td>61</td><td>62</td><td>37</td><td>77</td><td>53</td><td>74</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pru ar 1</td><td>64</td><td>61</td><td>18</td><td>46</td><td>50</td><td>60</td><td>59</td><td>64</td><td>44</td><td>73</td><td>51</td><td>73</td><td>72</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pru av 1</td><td>56</td><td>54</td><td>18</td><td>42</td><td>51</td><td>59</td><td>64</td><td>66</td><td>39</td><td>81</td><td>53</td><td>74</td><td>85</td><td>76</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pru p 1</td><td>57</td><td>55</td><td>18</td><td>42</td><td>50</td><td>59</td><td>65</td><td>64</td><td>40</td><td>83</td><td>53</td><td>74</td><td>83</td><td>77</td><td>98</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pyr c 1</td><td>52</td><td>50</td><td>20</td><td>39</td><td>50</td><td>57</td><td>60</td><td>62</td><td>38</td><td>76</td><td>52</td><td>75</td><td>94</td><td>73</td><td>84</td><td>84</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Rub i 1</td><td>52</td><td>57</td><td>15</td><td>43</td><td>47</td><td>55</td><td>59</td><td>62</td><td>40</td><td>69</td><td>48</td><td>71</td><td>68</td><td>72</td><td>72</td><td>72</td><td>68</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Sola l 4</td><td>48</td><td>48</td><td>19</td><td>39</td><td>41</td><td>44</td><td>49</td><td>50</td><td>35</td><td>49</td><td>43</td><td>51</td><td>44</td><td>51</td><td>49</td><td>50</td><td>43</td><td>47</td><td></td><td></td><td></td><td></td></tr><tr><td>Vig r 1</td><td>43</td><td>44</td><td>17</td><td>39</td><td>67</td><td>44</td><td>46</td><td>54</td><td>35</td><td>46</td><td>76</td><td>52</td><td>47</td><td>48</td><td>47</td><td>47</td><td>45</td><td>46</td><td>41</td><td></td><td></td><td></td></tr><tr><td>Vig r 6</td><td>23</td><td>23</td><td>15</td><td>20</td><td>20</td><td>26</td><td>25</td><td>19</td><td>18</td><td>24</td><td>24</td><td>25</td><td>23</td><td>25</td><td>22</td><td>23</td><td>22</td><td>22</td><td>24</td><td>23</td><td></td><td></td></tr></tbody><tfoot><tr><td colspan="24">Percent identity matrix was created using Clustal2.1 (EMBL-EBI)</td></tr></tfoot></table>

<table><caption>TABLE 78 Reported ligand classes interacting with PR-10 allergens</caption><thead><tr><th>Ligand chemical class</th><th>Reported allergens</th></tr></thead><tbody><tr><td>Animal steroid hormones</td><td>Ara h 8, Cor a 1, Que a 1</td></tr><tr><td>Brassinosteroid (analog)</td><td>Bet v 1, Cor a 1, Fag s 1, Pru av 1, Que a 1</td></tr><tr><td>Cytokinin</td><td>Cor a 1, Bet v 1, Fag s 1, Pru p 1</td></tr><tr><td>Extrinsic and non-physiological ligands</td><td>Bet v 1, Fag s 1</td></tr><tr><td>Fatty acids</td><td>Bet v 1, Cor a 1, Que a 1</td></tr><tr><td>Flavonoids</td><td>Ara h 8, Bet v 1, Cor a 1, Fag s 1, Fra a 1, Que a 1</td></tr><tr><td>Organic compounds</td><td>Ara h 8, Cor a 1, Fag s 1</td></tr><tr><td>Phytoprostanes</td><td>Bet v 1</td></tr></tbody></table>

the boys had IgE specific for birch pollen allergens. IgE sensitization to birch pollen allergens in adults (age 19 to 79 years) was found to be 17.4% and sensitization to Bet v 1 15.2%.1226 Approximately half of all sensitized individuals will develop symptoms such as allergic rhinoconjunctivitis or allergic asthma.798

According to the National Health and Nutrition Examination Survey (NHANES) 2005-2006 sensitization to birch pollen in the United States was on average 10% with slightly higher sensitization rates of 12.3% and 11.8% in the Northeast and Western regions, respectively.627 The NHANES 2005-2006 data also demonstrated racial/ethnic differences. Sensitization to birch pollen was significantly

higher in non-Hispanic blacks (14%) compared with non-Hispanic whites (9.1%).627

Oak, hazel and alder pollen account for sensitization to Bet v 1-homologous proteins in East Asia, with oak tree pollen dominating over pollen of other tree species due to climate change1446. The IgE-sensitization rate to oak pollen has more than doubled from 4.7% in 1998 to 9.8% in 2019 in children living in the Seoul metropolitan area.1446

### 3.3. Symptoms (respiratory allergy)

Typical mucosal symptoms of tree pollen allergy occur during springtime in the respective regions (in Central Europe between February and May) with maxima depending on the current climate and the region's altitude:

* itchiness, redness, tearing of the eyes

* itch in the nose, oropharyngeal itch, (repeated) sneezing, runny and/or stuffy nose

* occasionally dry cough (particularly during or shortly after exercise), dyspnea, pressure sensation on the chest, wheezing, bronchial secretion and difficulty in breathing as indicators for increasingly affected lower airways (asthmatic lower airway inflammation)

* occasionally flu-like symptoms such as fatigue, body aches and headaches

Clinical diagnoses of intermittent (seasonal) rhinoconjunctivitis and allergic asthma due to birch pollen become more likely when mucosal symptoms occur during the same season in subsequent
years. Additional oropharyngeal symptoms that occur in approximately 2/3 of birch pollen allergic individuals after consumption of certain raw plant foods indirectly confirm a suspected birch pollen allergy. In a minority of birch pollen allergic individuals, ingestion of processed plant foods such as roasted hazelnuts, products containing roasted hazelnuts, or cooked celeriac can induce OAS.

### 3.4. Symptoms (oral exposure)

Minute amounts of Bet v 1-homologous proteins can induce various, transient, predominantly oropharyngeal symptoms (Table 79) with a quick onset (sometimes immediately and often within minutes) after consumption of raw plant foods (Table 75, Figure 148) in approximately 2/3 of birch pollen allergic individuals. These symptoms are often referred to as "oral allergy syndrome" (OAS), implicating a particular clinical entity. This is not the case, since

* oropharyngeal symptoms can occur quite variably including different degrees of severity (Table 79 symptom complex A)

* diffusion of inflammatory mediators (i.e., histamine) and/or neutrogenic reflexes can prompt additional severe symptoms in the head area (Table 79 symptom complex B) or

* systemic, sometimes anaphylactic symptoms can occur in rare cases (Table 79 symptom complex C)

Noteworthy, oropharyngeal symptoms are not unique or specific for Bet v 1-induced cross-reactions or certain food items, since other food allergens are able to induce similar symptoms:

* profilin-containing plant foods (see chapter C01)

* nonspecific lipid transfer protein (nsLTP)-containing plant foods (see chapter C03)

* gibberellin-regulated protein (GRP)-containing plant foods (see chapter C09)

* seed storage proteins-containing plant foods (see chapter C08)

* cystein protease-containing plant foods

* β-1,3-glucanase-containing plant foods

* animal derived food allergens (see chapters B10-B14)

Thus, the so-called OAS does not represent a defined clinical entity (syndrome) but a rather variable symptom complex. The occurrence of only oropharyngeal symptoms reflects the physicochemical properties of the particular food allergens, which in the case of the Bet v 1-homologous proteins are

* instability, i.e., poor resistance to degradation by digestive proteolytic enzymes and consequently rarely a substantial intestinal absorption

* excellent aqueous solubility (quick onset of symptoms after mucosal exposure).

From a clinical point of view, a large and over the years increasing number of reported Bet v 1-related cross-reactive plant foods indicates a high level of Bet v 1-specific IgE based on a broad polyclonal IgE repertoire.

The following variables are presumably involved in rare, severe clinical reactions to foods containing Bet v 1-homologues:

* a strong Bet v 1-specific IgE response (high specific IgE/total IgE ratio)

* a broad Bet v 1-specific IgE repertoire (indirectly reflected in a large panel of implicated plant foods)

* the ingested amount of a particular Bet v 1-homologue containing food

* ingestion of a Bet v 1-homologue-containing food on an empty stomach

<table><caption>TABLE 79 Potential symptoms due to Bet v 1-associated IgE cross-reactivity to plant foods</caption><thead><tr><th>Symptom complex</th><th>Symptoms</th><th>Organ/localization</th></tr></thead><tbody><tr><td rowspan="4">A. Limited oropharyngeal symptoms (frequent)</td><td>itch ("tingling", "tickling", "prickle")</td><td>lip mucosa, oral mucosa, palate</td></tr><tr><td>burning, stinging</td><td>palate, throat</td></tr><tr><td>mild mucosal swelling</td><td>lip mucosa, oral mucosa, palate, throat</td></tr><tr><td>itch, redness, tearing</td><td>conjunctiva</td></tr><tr><td rowspan="5">B. Additional symptoms in the head area (isolated or with symptoms from A) (rare events)</td><td>itch, sneezing, runny nose, stuffy nose</td><td>nose</td></tr><tr><td>itch</td><td>ears (internally, Eustachian tubes)</td></tr><tr><td>cutaneous and subcutaneous swelling (urticaria, angioedema)</td><td>eye lids, lips, cheeks, ears, face</td></tr><tr><td>severe (internal) mucosal swelling, globus sensation, difficulty swallowing, hoarseness (indicating vocal cord or larynx edema), shortness of breath, stridor</td><td>palate, throat, larynx</td></tr><tr><td>itch, redness, hives, swelling (urticaria, angioedema)</td><td>localized, multifocal or generalized at the skin</td></tr><tr><td rowspan="4">C. Systemic symptoms (extremely rare)</td><td>nausea, vomiting, abdominal pain, diarrhea</td><td>stomach, intestine</td></tr><tr><td>breathing difficulties, chest pressure, chest tightness, shortness of breath, wheezing, cough, sputum (optional)</td><td>bronchi</td></tr><tr><td>dizziness, general weakness, unconsciousness, circulatory collapse</td><td>heart circulation</td></tr></tbody></table>
years. Additional oropharyngeal symptoms that occur in approximately 2/3 of birch pollen allergic individuals after consumption of certain raw plant foods indirectly confirm a suspected birch pollen allergy. In a minority of birch pollen allergic individuals, ingestion of processed plant foods such as roasted hazelnuts, products containing roasted hazelnuts, or cooked celeriac can induce OAS.

### 3.4. Symptoms (oral exposure)

Minute amounts of Bet v 1-homologous proteins can induce various, transient, predominantly oropharyngeal symptoms (Table 79) with a quick onset (sometimes immediately and often within minutes) after consumption of raw plant foods (Table 75, Figure 148) in approximately 2/3 of birch pollen allergic individuals. These symptoms are often referred to as "oral allergy syndrome" (OAS), implicating a particular clinical entity. This is not the case, since

* oropharyngeal symptoms can occur quite variably including different degrees of severity (Table 79 symptom complex A)

* diffusion of inflammatory mediators (i.e., histamine) and/or neutrogenic reflexes can prompt additional severe symptoms in the head area (Table 79 symptom complex B) or

* systemic, sometimes anaphylactic symptoms can occur in rare cases (Table 79 symptom complex C)

Noteworthy, oropharyngeal symptoms are not unique or specific for Bet v 1-induced cross-reactions or certain food items, since other food allergens are able to induce similar symptoms:

* profilin-containing plant foods (see chapter C01)

* nonspecific lipid transfer protein (nsLTP)-containing plant foods (see chapter C03)

* gibberellin-regulated protein (GRP)-containing plant foods (see chapter C09)

* seed storage proteins-containing plant foods (see chapter C08)

* cystein protease-containing plant foods

* β-1,3-glucanase-containing plant foods

* animal derived food allergens (see chapters B10-B14)

Thus, the so-called OAS does not represent a defined clinical entity (syndrome) but a rather variable symptom complex. The occurrence of only oropharyngeal symptoms reflects the physicochemical properties of the particular food allergens, which in the case of the Bet v 1-homologous proteins are

* instability, i.e., poor resistance to degradation by digestive proteolytic enzymes and consequently rarely a substantial intestinal absorption

* excellent aqueous solubility (quick onset of symptoms after mucosal exposure).

From a clinical point of view, a large and over the years increasing number of reported Bet v 1-related cross-reactive plant foods indicates a high level of Bet v 1-specific IgE based on a broad polyclonal IgE repertoire.

The following variables are presumably involved in rare, severe clinical reactions to foods containing Bet v 1-homologues:

* a strong Bet v 1-specific IgE response (high specific IgE/total IgE ratio)

* a broad Bet v 1-specific IgE repertoire (indirectly reflected in a large panel of implicated plant foods)

* the ingested amount of a particular Bet v 1-homologue containing food

* ingestion of a Bet v 1-homologue-containing food on an empty stomach

<table><caption>TABLE 79 Potential symptoms due to Bet v 1-associated IgE cross-reactivity to plant foods</caption><thead><tr><th>Symptom complex</th><th>Symptoms</th><th>Organ/localization</th></tr></thead><tbody><tr><td rowspan="4">A. Limited oropharyngeal symptoms (frequent)</td><td>itch ("tingling", "tickling", "prickle")</td><td>lip mucosa, oral mucosa, palate</td></tr><tr><td>burning, stinging</td><td>palate, throat</td></tr><tr><td>mild mucosal swelling</td><td>lip mucosa, oral mucosa, palate, throat</td></tr><tr><td>itch, redness, tearing</td><td>conjunctiva</td></tr><tr><td rowspan="5">B. Additional symptoms in the head area (isolated or with symptoms from A) (rare events)</td><td>itch, sneezing, runny nose, stuffy nose</td><td>nose</td></tr><tr><td>itch</td><td>ears (internally, Eustachian tubes)</td></tr><tr><td>cutaneous and subcutaneous swelling (urticaria, angioedema)</td><td>eye lids, lips, cheeks, ears, face</td></tr><tr><td>severe (internal) mucosal swelling, globus sensation, difficulty swallowing, hoarseness (indicating vocal cord or larynx edema), shortness of breath, stridor</td><td>palate, throat, larynx</td></tr><tr><td>itch, redness, hives, swelling (urticaria, angioedema)</td><td>localized, multifocal or generalized at the skin</td></tr><tr><td rowspan="4">C. Systemic symptoms (extremely rare)</td><td>nausea, vomiting, abdominal pain, diarrhea</td><td>stomach, intestine</td></tr><tr><td>breathing difficulties, chest pressure, chest tightness, shortness of breath, wheezing, cough, sputum (optional)</td><td>bronchi</td></tr><tr><td>dizziness, general weakness, unconsciousness, circulatory collapse</td><td>heart circulation</td></tr></tbody></table>
* differences in stability of the Bet v 1-homologue in the respective food items (more often systemic reactions due to hazelnuts, soy, carrots and celeriac in comparison to e.g., apples)

* matrix effects of certain food items (e.g., soy) with high protein content, protecting the immediate degradation of a Bet v 1-homologue-containing food.

An increase in oropharyngeal symptoms during or shortly after the birch pollen season is generally observed. Natural birch pollen exposure might boost the Bet v 1-specific IgE response resulting in a broadened IgE repertoire.

Occasionally, patients experience oropharyngeal symptoms (Table 79) after consumption of typical Bet v 1-related plant foods without suffering from inhalant symptoms during the tree pollen season. This observation, a (so far) clinically "silent" Bet v 1 sensitization, can still prompt unexpected allergic reactions after the first consumption of Bet v 1-related plant foods. Without knowledge of the molecular relationship both, the diagnosis of a birch pollen (Bet v 1)-associated plant food allergy, and proper consultation of the affected individuals might be unnecessarily delayed.

The most frequently observed allergy to soy in Central Europe is based on the serological cross-reactivity of Bet v 1-specific IgE (70%) with its homologue Gly m 4 from soybean (see chapter B17). In general, reactions occur following the ingestion of large amounts of soy-based products that did not undergo major processing steps (e.g., soymilk, soy-based protein powders). Reactions can be systemic and severe and have been observed in about 10% of birch-pollen allergic individuals.<sup>1217</sup>

## Conclusions on clinical relevance

* Sensitization to the PR-10-like allergen Bet v 1 and its homologous proteins in pollen from Fagales tree species occurs worldwide except in South Africa and the tropics.

* Around half of all sensitized individuals will develop symptoms.

* Around 70% of birch pollen allergic individuals suffer from associated plant food allergy.

* Impact of IgE-cross-reactivity on clinical symptoms of food allergy is still unknown.

* Molecule-based therapeutic approaches are under investigation.

# 4 - Clinical management

## Diagnostic testing of Bet v 1-related allergies

IgE sensitization to Bet v 1 can be assessed (Figure 149) directly by allergen-specific IgE testing to Bet v 1, or indirectly by SPT with birch pollen extract or allergen-specific IgE testing with birch

pollen extract when sensitization to Bet v 2 has been excluded by allergen-specific IgE testing. Convincing analytical specificity after birch pollen extract testing is only obtained in case of a limited sensitization profile (i.e., if only positive to pollen of Fagales trees). If a positive SPT result was already obtained with a birch pollen extract, allergen-specific IgE should rather be tested to Bet v 1, providing more (analytical) specificity than another birch pollen extract for in vitro diagnosis.

Commercial plant food extracts (for SPT and IgE testing) often show false-negative results and should be avoided due to a low abundance and a lack of stability of Bet v 1 homologous food allergens. Instead, prick-to-prick tests can be performed with suspected fresh, raw plant foods. Despite a lack of standardization, this approach can provide a qualitative, indirect demonstration of IgE sensitization.

## No value of broad molecular testing of Bet v 1-sensitized patients

Is it worth it to demonstrate additional IgE sensitizations/cross-reactions to Bet v 1-homologues such as allergen-specific IgE to Mal d 1, Cor a 1.04, Pru p 1 and many others? It is not, since numerous positive serological cross-reactions are to be expected when Bet v 1-specific IgE is present.<sup>1447</sup> Thus, such test results are not informative in terms of their clinical relevance without further clinical information. Only a clear-cut negative IgE result obtained for an individual Bet v 1-homologous allergen (i.e., Dau c 1, Gly m 4, Pru p 1), obtained with a sensitive IgE test (cut-off at 0.1 kU/L; singleplex assay) could reliably rule out an IgE sensitization/cross-reactivity and a subsequent clinically relevant food allergy due to Bet v 1-cross-reactivity. Unfortunately, this scenario does hardly exist. Instead, rational and targeted testing (i.e., allergen-specific IgE only to Bet v 1) is strongly recommended. Sensitization tests in case of Bet v 1-associated cross-reactions are only meaningful, if the results will potentially lead to clinical consequences.

## Interpretation of Bet v 1-related IgE sensitizations

Interpretation of a test addressing the clinical relevance of a previously demonstrated Bet v 1-sensitization can only be obtained by taking into account the patient's clinical history. Thus, a detailed anamnesis is very important in case a birch pollen-associated food allergy is in question. The following suggested diagnostic work-up can facilitate a final interpretation of the clinical relevance of a sensitization test (i.e., positive birch pollen SPT, positive Bet v 1-specific IgE):

* The subject should be systematically asked, whether oropharyngeal (or other) allergic symptoms (Table 79) occurred and after consumption of which raw plant foods these symptoms appeared.

* The list of foods should not only address the most common ones as apples and hazelnuts, but the whole panel of potentially Bet v 1-cross-reactive plant foods (Table 75, Figure 148). Oral challenge tests can help—in case of missing clinical information or vague anamnesis
* differences in stability of the Bet v 1-homologue in the respective food items (more often systemic reactions due to hazelnuts, soy, carrots and celeriac in comparison to e.g., apples)

* matrix effects of certain food items (e.g., soy) with high protein content, protecting the immediate degradation of a Bet v 1-homologue-containing food.

An increase in oropharyngeal symptoms during or shortly after the birch pollen season is generally observed. Natural birch pollen exposure might boost the Bet v 1-specific IgE response resulting in a broadened IgE repertoire.

Occasionally, patients experience oropharyngeal symptoms (Table 79) after consumption of typical Bet v 1-related plant foods without suffering from inhalant symptoms during the tree pollen season. This observation, a (so far) clinically "silent" Bet v 1 sensitization, can still prompt unexpected allergic reactions after the first consumption of Bet v 1-related plant foods. Without knowledge of the molecular relationship both, the diagnosis of a birch pollen (Bet v 1)-associated plant food allergy, and proper consultation of the affected individuals might be unnecessarily delayed.

The most frequently observed allergy to soy in Central Europe is based on the serological cross-reactivity of Bet v 1-specific IgE (70%) with its homologue Gly m 4 from soybean (see chapter B17). In general, reactions occur following the ingestion of large amounts of soy-based products that did not undergo major processing steps (e.g., soymilk, soy-based protein powders). Reactions can be systemic and severe and have been observed in about 10% of birch-pollen allergic individuals.<sup>1217</sup>

## Conclusions on clinical relevance

* Sensitization to the PR-10-like allergen Bet v 1 and its homologous proteins in pollen from Fagales tree species occurs worldwide except in South Africa and the tropics.

* Around half of all sensitized individuals will develop symptoms.

* Around 70% of birch pollen allergic individuals suffer from associated plant food allergy.

* Impact of IgE-cross-reactivity on clinical symptoms of food allergy is still unknown.

* Molecule-based therapeutic approaches are under investigation.

# 4 - Clinical management

## Diagnostic testing of Bet v 1-related allergies

IgE sensitization to Bet v 1 can be assessed (Figure 149) directly by allergen-specific IgE testing to Bet v 1, or indirectly by SPT with birch pollen extract or allergen-specific IgE testing with birch

pollen extract when sensitization to Bet v 2 has been excluded by allergen-specific IgE testing. Convincing analytical specificity after birch pollen extract testing is only obtained in case of a limited sensitization profile (i.e., if only positive to pollen of Fagales trees). If a positive SPT result was already obtained with a birch pollen extract, allergen-specific IgE should rather be tested to Bet v 1, providing more (analytical) specificity than another birch pollen extract for in vitro diagnosis.

Commercial plant food extracts (for SPT and IgE testing) often show false-negative results and should be avoided due to a low abundance and a lack of stability of Bet v 1 homologous food allergens. Instead, prick-to-prick tests can be performed with suspected fresh, raw plant foods. Despite a lack of standardization, this approach can provide a qualitative, indirect demonstration of IgE sensitization.

## No value of broad molecular testing of Bet v 1-sensitized patients

Is it worth it to demonstrate additional IgE sensitizations/cross-reactions to Bet v 1-homologues such as allergen-specific IgE to Mal d 1, Cor a 1.04, Pru p 1 and many others? It is not, since numerous positive serological cross-reactions are to be expected when Bet v 1-specific IgE is present.<sup>1447</sup> Thus, such test results are not informative in terms of their clinical relevance without further clinical information. Only a clear-cut negative IgE result obtained for an individual Bet v 1-homologous allergen (i.e., Dau c 1, Gly m 4, Pru p 1), obtained with a sensitive IgE test (cut-off at 0.1 kU/L; singleplex assay) could reliably rule out an IgE sensitization/cross-reactivity and a subsequent clinically relevant food allergy due to Bet v 1-cross-reactivity. Unfortunately, this scenario does hardly exist. Instead, rational and targeted testing (i.e., allergen-specific IgE only to Bet v 1) is strongly recommended. Sensitization tests in case of Bet v 1-associated cross-reactions are only meaningful, if the results will potentially lead to clinical consequences.

## Interpretation of Bet v 1-related IgE sensitizations

Interpretation of a test addressing the clinical relevance of a previously demonstrated Bet v 1-sensitization can only be obtained by taking into account the patient's clinical history. Thus, a detailed anamnesis is very important in case a birch pollen-associated food allergy is in question. The following suggested diagnostic work-up can facilitate a final interpretation of the clinical relevance of a sensitization test (i.e., positive birch pollen SPT, positive Bet v 1-specific IgE):

* The subject should be systematically asked, whether oropharyngeal (or other) allergic symptoms (Table 79) occurred and after consumption of which raw plant foods these symptoms appeared.

* The list of foods should not only address the most common ones as apples and hazelnuts, but the whole panel of potentially Bet v 1-cross-reactive plant foods (Table 75, Figure 148). Oral challenge tests can help—in case of missing clinical information or vague anamnesis
<figure><img src="image_272.png" /><figcaption>FIGURE 149 Diagnostic algorithm in case of Fagales tree pollen and/or Bet v 1 homologue-related food allergy to raw plant foods. *If the patient tolerates the processed food, only the unprocessed food should be avoided.</figcaption></figure>

* to prove or rule out clinical cross-reactions. They can also serve as a prospective test of a potentially cross-reactive, but so far never eaten plant food.

Oral food challenges, however, are rarely routinely performed in case of Bet v 1-associated food allergies for the following reasons:

* There is no urgent indication in case of solely oropharyngeal symptoms.

* They are not easy to interpret due to predominantly subjective symptoms.

* There are almost no validated dose-dependent tests.

* There are only a few proven published protocols for oral challenge procedures with Bet v 1-associated plant foods.¹⁴⁴⁸

* They are tedious to perform considering the number of potentially cross-reactive foods.

It is of utmost importance to advise patients that only those Bet v 1-related foods should be avoided in their raw form, when they have induced typical allergic symptoms. Avoiding every potentially cross-reactive food that may contain a Bet v 1-homologous allergen is clinically not justified and would be exaggerated. The same is true for plant foods, showing indirectly after prick-to-prick tests or directly after serological analysis positive IgE sensitizations. Even the entire panel of Bet v 1-homologues employed for diagnostic purposes would not be able to separate silent from clinically relevant sensitizations in case of positive test results.

### Diagnostic recommendations for Bet v 1-related allergy

* Bet v 1-homologues in tree pollen extracts for diagnostic purposes will induce positive SPT and IgE reactions to pollen of several Fagales tree (Table 74, Figure 147) not necessarily being clinically relevant.

* Prick-to-Prick tests with fresh (raw) foods are superior diagnostic tools compared with commercial food extracts in case of birch pollen-associated plant food allergies due to low stability of Bet v 1-homologues.

* After being spiked with recombinant Bet v 1-homologues (e.g., Cora 1) birch pollen-associated plant food extracts (i.e., hazelnut extract) can bind more IgE, increase assay sensitivity (lowered "limit of quantitation," LoQ) and provide elevated allergen-specific IgE values.

* At the same time "spiking" will unfold more positive (potentially clinically irrelevant) sensitizations, pointing to general drawbacks of extract-based diagnostics (i.e., high sensitization rates to peanut in Central Europe due to cross-reactive natural Bet v 1-homologue Ara h 8 in peanut extracts).

* Bet v 1-specific IgE serves as a reliable marker for potential, serological cross-reactions to a number of plant foods (Table 75, Figure 148). The clinical relevance of potential cross-reactions is systematically addressed by the physician together with the patient based on the subject's individual symptoms after consumption of raw foods containing Bet v 1-homologues.

* Positive specific IgE to Bet v 1-homologues plant food allergens (i.e., Pru p 1 from peach) demonstrates allergic sensitization, being only clinically relevant in case of corresponding symptoms.

* A negative IgE results (i.e., to Gly m 4 from soy, approx. in only 25% of Bet v 1-sensitized subjects) would exclude serological cross-reactivity with certainty and clinically relevant symptomatic cross-reactions as well.

The rule-of-thumb for the diagnostic work-up of Bet v 1-associated allergic reactions is: "The physician's interpretation, based on the patient's individual symptoms, and not the outcome of a sensitization test will establish the decision about the clinical relevance of previous diagnostic findings (personal comment by author JKT).
<figure><img src="image_278.png" /><figcaption>FIGURE 149 Diagnostic algorithm in case of Fagales tree pollen and/or Bet v 1 homologue-related food allergy to raw plant foods. *If the patient tolerates the processed food, only the unprocessed food should be avoided.</figcaption></figure>

* to prove or rule out clinical cross-reactions. They can also serve as a prospective test of a potentially cross-reactive, but so far never eaten plant food.

Oral food challenges, however, are rarely routinely performed in case of Bet v 1-associated food allergies for the following reasons:

* There is no urgent indication in case of solely oropharyngeal symptoms.

* They are not easy to interpret due to predominantly subjective symptoms.

* There are almost no validated dose-dependent tests.

* There are only a few proven published protocols for oral challenge procedures with Bet v 1-associated plant foods.<sup>1448</sup>

* They are tedious to perform considering the number of potentially cross-reactive foods.

It is of utmost importance to advise patients that only those Bet v 1-related foods should be avoided in their raw form, when they have induced typical allergic symptoms. Avoiding every potentially cross-reactive food that may contain a Bet v 1-homologous allergen is clinically not justified and would be exaggerated. The same is true for plant foods, showing indirectly after prick-to-prick tests or directly after serological analysis positive IgE sensitizations. Even the entire panel of Bet v 1-homologues employed for diagnostic purposes would not be able to separate silent from clinically relevant sensitizations in case of positive test results.

### Diagnostic recommendations for Bet v 1-related allergy

* Bet v 1-homologues in tree pollen extracts for diagnostic purposes will induce positive SPT and IgE reactions to pollen of several Fagales tree (Table 74, Figure 147) not necessarily being clinically relevant.

* Prick-to-Prick tests with fresh (raw) foods are superior diagnostic tools compared with commercial food extracts in case of birch pollen-associated plant food allergies due to low stability of Bet v 1-homologues.

* After being spiked with recombinant Bet v 1-homologues (e.g., Cora 1) birch pollen-associated plant food extracts (i.e., hazelnut extract) can bind more IgE, increase assay sensitivity (lowered "limit of quantitation," LoQ) and provide elevated allergen-specific IgE values.

* At the same time "spiking" will unfold more positive (potentially clinically irrelevant) sensitizations, pointing to general drawbacks of extract-based diagnostics (i.e., high sensitization rates to peanut in Central Europe due to cross-reactive natural Bet v 1-homologue Ara h 8 in peanut extracts).

* Bet v 1-specific IgE serves as a reliable marker for potential, serological cross-reactions to a number of plant foods (Table 75, Figure 148). The clinical relevance of potential cross-reactions is systematically addressed by the physician together with the patient based on the subject's individual symptoms after consumption of raw foods containing Bet v 1-homologues.

* Positive specific IgE to Bet v 1-homologues plant food allergens (i.e., Pru p 1 from peach) demonstrates allergic sensitization, being only clinically relevant in case of corresponding symptoms.

* A negative IgE results (i.e., to Gly m 4 from soy, approx. in only 25% of Bet v 1-sensitized subjects) would exclude serological cross-reactivity with certainty and clinically relevant symptomatic cross-reactions as well.

The rule-of-thumb for the diagnostic work-up of Bet v 1-associated allergic reactions is: "The physician's interpretation, based on the patient's individual symptoms, and not the outcome of a sensitization test will establish the decision about the clinical relevance of previous diagnostic findings (personal comment by author JKT).
Sensitization can be tested by skin prick testing or in vitro by extract- or molecule-based assays. IgE test results must be interpreted always in the context of the anamnesis. At present, all Bet v 1-related allergens that are of broader clinical relevance are available as recombinant proteins but only a few for diagnostic purposes.

## Extract composition for allergen-specific immunotherapy

Due to the high cross-reactivity of the major allergens Bet v 1, Cor a 1, Que a 1 and Aln g 1 of birch, hazel, oak and alder pollen, respectively, birch pollen mono-extracts are suitable for specific immunotherapy of a tree pollen allergy.⁶¹⁶ Whether immunotherapy with tree pollen extracts has a beneficial influence on associated plant food allergies is still under discussion. Most studies were performed on birch pollen-associated apple allergy. As the apple allergen Mal d 1 has a very high sequence identity to Bet v 1, one might expect very good results from a birch pollen-based immunotherapy. However, the results from several studies on birch pollen-associated apple allergy are controversial.¹¹⁷²,¹¹⁷³,¹⁶³⁶ Likewise, no clinical effect on a birch pollen-associated hazelnut allergy was observed after one year of specific immunotherapy with a birch pollen extract.¹⁶³⁷

Therefore, an oral allergy-syndrome to birch pollen-associated plant foods in the absence of pollen induced respiratory symptoms should not be considered as a main criterion for selecting patients for birch pollen immunotherapy. Whether immunotherapy with Bet v 1-homologous plant food allergens might be an option for patients with severe oral allergy symptoms has to be studied in larger clinical trials. Data from phase II studies have shown that sublingual immunotherapy with recombinant Mal d 1 can effectively reduce oral allergy symptoms in apple-allergic patients,⁶⁵ and oral immunotherapy with raw apple significantly increased increased Mal d 1-specific IgG4 and tolerance to apples¹⁴⁴⁴

# 5 - Clinical cases

## Case 1 (original, #6166):

**History:** Female, 39 yrs: Since 2015 for the first time during spring time eye itch, tearing, swelling, sneezing, runny and blocked nose, later chest tightness, wheezing, coughing and white sputum. In addition, since spring 2015 after raw fruits (apples, cherries, peaches) itchy throat.

**In-vivo testing:** SPT (wheal diameter [mm]): hazel 12, alder 5, birch 4, oak 6.

**In-vitro testing:** Total IgE 190 kU/L, specific IgE to Bet v 1: 91 kU/L (>47% of total IgE indicating a strong sensitization).

**Diagnosis:** (A) Allergic rhinoconjunctivitis due to Fagales tree pollen; (B) Bet v 1-associated food allergy (oropharyngeal symptoms to certain raw Rosaceae fruits). Recommendations: Allergen-specific immunotherapy with birch pollen extract with caution during the escalation phase (high relative specific IgE to Bet v 1 is associated with potential adverse events). Avoidance of raw Bet v 1-cross-reactive pome and stone fruits (see history); cooked, baked or roasted plant products without dietary restriction (due to thermal instability of Bet v 1-related allergens).

**Comments:** (a) Strong Bet v 1-sensitization based on high absolute IgE values and high ratio between Bet v 1-specific IgE and total IgE; (b) despite the short allergy history (first allergy season!) strong indication for AIT due to the rapid disease development including lower airways and cross-reactive oropharyngeal symptoms.

## Case 2 (original, #6112)

**History:** Male, 35 yrs: Since 10 years Fagales tree pollen-induced rhinoconjunctivitis with itchy eyes, sneezing, runny nose, sore throat, itch in the ears, general fatigue. After ingestion of raw apples, hazelnuts, cherries, peaches, apricots, strawberries, blueberries*, grapes* within 5 minutes itchy and sore throat, itchy eyes and ears for 15 minutes, after soy products loose stool.

**In vivo testing:** SPT (wheal diameter [mm]): hazel 6, alder 5, birch 10, oak 3, grass-mix 3, mugwort 3.

**In vitro testing:** Total IgE 10 kU/L, specific IgE to Bet v 1: 3.4 kU/L (>1/3 of total IgE indicating strong sensitization), Phl p 1: 2.8 kU/L, other allergen specificities Phl p 12 (profilin), Art v 1, mugwort, Pru p 3 (nsLTP) negative (<0.1 kU/L).

**Diagnosis:** (A) Allergic rhinoconjunctivitis due to Fagales tree pollen; (B) Bet v 1-associated food allergy (oropharyngeal symptoms to raw foods). Recommendations: Allergen-specific immunotherapy with birch extract. Avoidance of raw Bet v 1-cross-reactive pome and stone fruits (see subject's history).

**Comments:** (a) Absolute IgE values (total and allergen-specific) are both low; (b) relative relationship between Bet v 1-specific IgE and total IgE is high (pointing to the need to consider both, total and specific IgE, for proper interpretation); (c) *certain fruits are not primarily regarded as containing Bet v 1-homologues, but rather as nsLTP-containing foods; but, due to negative IgE to Pru p 3 and profilin, these reported reactions cannot be easily explained. A Bet v 1 homologue may well be described in blueberries and grapes in the future.

## Case 3 (original, #6213):

**History:** Female, 47 yrs: Since 15 years after consumption of raw plant foods like apples, cherries, hazelnuts, walnuts within 1 minute mild sore throat for 5 minutes. So far, no allergic symptoms during spring.

**In vitro testing:** Total IgE 174 kU/L, specific IgE to Bet v 1: 34 kU/L.

**Diagnosis:** Bet v 1-associated food allergy (oropharyngeal symptoms to raw foods) without allergic airway disease. Recommendations: Avoidance of raw Bet v 1-cross-reactive pome and stone fruits (see subject's history). No indication for AIT due to missing Fagales tree pollen induced airway symptoms.

**Comments:** Rare cases are suffering from oropharyngeal allergy symptoms due to plant foods containing Bet v 1-homologues without any allergic airway symptoms during the birch pollen season. Diagnosis is established by IgE testing to Bet v 1. Potential clinical consequences are addressed "clinically" (without further IgE testing to other Bet v 1-homologues).
Sensitization can be tested by skin prick testing or *in vitro* by extract- or molecule-based assays. IgE test results must be interpreted always in the context of the anamnesis. At present, all Bet v 1-related allergens that are of broader clinical relevance are available as recombinant proteins but only a few for diagnostic purposes.

### Extract composition for allergen-specific immunotherapy

Due to the high cross-reactivity of the major allergens Bet v 1, Cor a 1, Que a 1 and Aln g 1 of birch, hazel, oak and alder pollen, respectively, birch pollen mono-extracts are suitable for specific immunotherapy of a tree pollen allergy.⁶¹⁶ Whether immunotherapy with tree pollen extracts has a beneficial influence on associated plant food allergies is still under discussion. Most studies were performed on birch pollen-associated apple allergy. As the apple allergen Mal d 1 has a very high sequence identity to Bet v 1, one might expect very good results from a birch pollen-based immunotherapy. However, the results from several studies on birch pollen-associated apple allergy are controversial.¹¹⁷²,¹¹⁷³,¹⁶³⁶ Likewise, no clinical effect on a birch pollen-associated hazelnut allergy was observed after one year of specific immunotherapy with a birch pollen extract.¹⁶³⁷

Therefore, an oral allergy-syndrome to birch pollen-associated plant foods in the absence of pollen induced respiratory symptoms should not be considered as a main criterion for selecting patients for birch pollen immunotherapy. Whether immunotherapy with Bet v 1-homologous plant food allergens might be an option for patients with severe oral allergy symptoms has to be studied in larger clinical trials. Data from phase II studies have shown that sublingual immunotherapy with recombinant Mal d 1 can effectively reduce oral allergy symptoms in apple-allergic patients,⁶⁵ and oral immunotherapy with raw apple significantly increased increased Mal d 1-specific IgG4 and tolerance to apples¹⁴⁴⁴

## 5 - Clinical cases

### Case 1 (original, #6166):

**History:** Female, 39 yrs: Since 2015 for the first time during spring time eye itch, tearing, swelling, sneezing, runny and blocked nose, later chest tightness, wheezing, coughing and white sputum. In addition, since spring 2015 after raw fruits (apples, cherries, peaches) itchy throat.

**In-vivo testing:** SPT (wheal diameter [mm]): hazel 12, alder 5, birch 4, oak 6.

**In-vitro testing:** Total IgE 190 kU/L, specific IgE to Bet v 1: 91 kU/L (>47% of total IgE indicating a strong sensitization).

**Diagnosis:** (A) Allergic rhinoconjunctivitis due to Fagales tree pollen; (B) Bet v 1-associated food allergy (oropharyngeal symptoms to certain raw Rosaceae fruits). Recommendations: Allergen-specific immunotherapy with birch pollen extract with caution during the escalation phase (high relative specific IgE to Bet v 1 is associated with potential adverse events). Avoidance of raw Bet v 1-cross-reactive pome and stone fruits (see history); cooked, baked or roasted plant products without dietary restriction (due to thermal instability of Bet v 1-related allergens).

**Comments:** (a) Strong Bet v 1-sensitization based on high absolute IgE values and high ratio between Bet v 1-specific IgE and total IgE; (b) despite the short allergy history (first allergy season!) strong indication for AIT due to the rapid disease development including lower airways and cross-reactive oropharyngeal symptoms.

### Case 2 (original, #6112)

**History:** Male, 35 yrs: Since 10 years Fagales tree pollen-induced rhinoconjunctivitis with itchy eyes, sneezing, runny nose, sore throat, itch in the ears, general fatigue. After ingestion of raw apples, hazelnuts, cherries, peaches, apricots, strawberries, blueberries*, grapes* within 5 minutes itchy and sore throat, itchy eyes and ears for 15 minutes, after soy products loose stool.

**In vivo testing:** SPT (wheal diameter [mm]): hazel 6, alder 5, birch 10, oak 3, grass-mix 3, mugwort 3.

**In vitro testing:** Total IgE 10 kU/L, specific IgE to Bet v 1: 3.4 kU/L (>1/3 of total IgE indicating strong sensitization), Phl p 1: 2.8 kU/L, other allergen specificities Phl p 12 (profilin), Art v 1, mugwort, Pru p 3 (nsLTP) negative (<0.1 kU/L).

**Diagnosis:** (A) Allergic rhinoconjunctivitis due to Fagales tree pollen; (B) Bet v 1-associated food allergy (oropharyngeal symptoms to raw foods). Recommendations: Allergen-specific immunotherapy with birch extract. Avoidance of raw Bet v 1-cross-reactive pome and stone fruits (see subject's history).

**Comments:** (a) Absolute IgE values (total and allergen-specific) are both low; (b) relative relationship between Bet v 1-specific IgE and total IgE is high (pointing to the need to consider both, total and specific IgE, for proper interpretation); (c) *certain fruits are not primarily regarded as containing Bet v 1-homologues, but rather as nsLTP-containing foods; but, due to negative IgE to Pru p 3 and profilin, these reported reactions cannot be easily explained. A Bet v 1 homologue may well be described in blueberries and grapes in the future.

### Case 3 (original, #6213):

**History:** Female, 47 yrs: Since 15 years after consumption of raw plant foods like apples, cherries, hazelnuts, walnuts within 1 minute mild sore throat for 5 minutes. So far, no allergic symptoms during spring.

**In vitro testing:** Total IgE 174 kU/L, specific IgE to Bet v 1: 34 kU/L.

**Diagnosis:** Bet v 1-associated food allergy (oropharyngeal symptoms to raw foods) without allergic airway disease. Recommendations: Avoidance of raw Bet v 1-cross-reactive pome and stone fruits (see subject's history). No indication for AIT due to missing Fagales tree pollen induced airway symptoms.

**Comments:** Rare cases are suffering from oropharyngeal allergy symptoms due to plant foods containing Bet v 1-homologues without any allergic airway symptoms during the birch pollen season. Diagnosis is established by IgE testing to Bet v 1. Potential clinical consequences are addressed "clinically" (without further IgE testing to other Bet v 1-homologues).
# C03 – Nonspecific lipid transfer proteins (nsLTP)

Elide Anna Pastorello, Francisca Gomez, Domingo Barber

## Highlights

* Nonspecific lipid transfer proteins (nsLTP) are the most prevalent plant-food allergens in Southern Europe.

* The clinical reactions can be systemic and severe, especially when not associated to birch pollinosis.

* Pru p 3, the major allergen of peach, plays a precursor role in the sensitization to other nsLTP.

* Relevant nsLTP containing plant-foods belong not only to the *Rosaceae* family but also to the nut group and to cereals, such as wheat, maize, and rice.

## 1 – The protein

Pru p 3, the major allergen of peach, was the first nsLTP to be fully identified and characterized as a relevant food allergen. It is the most broadly recognized allergen of the family; however, there are patients sensitized to nsLTP that might not be sensitized to Pru p 3. It is in vascular tissue and in the outer cell layers of the plant organs and it essentially concentrates in the pericarp of fruits, whereas the pulp contains levels around 220-fold lower than peel. Peach fuzz contains large amounts of Pru p 3. It is a small basic protein of 91 amino acids, with a molecular weight of 9,178 Da. It contains a highly conserved domain consisting of alpha-helices that harbor eight cysteine residues, a distinctive sign of belonging to the Prolamin superfamily. The cysteine residues form four intramolecular disulphide bridges, which makes the protein resistant to high temperature and pH changes. The four disulphide bridges are responsible for the LTP compact folding, which forms a tunnel-like hydrophobic cavity running through the whole molecule. The Pru p 3 molecule is very flexible; therefore, the volume of the inner cavity can change significantly, and the protein can bind various lipidic ligands. The changes of the allergen conformation, which are related to ligand binding, alter the protein surface and this can modify its IgE-binding properties. This has been proposed for Pru p 3, where using an in silico approach, only the binding with oleic acid and not with stearic acid conditioned the exposure of the C-terminal loop that is a major IgE-binding epitope thus increasing the IgE-binding properties of this molecule.⁴⁸² Moreover, the natural ligand of Pru p 3 has been recently identified as a derivative of the alkaloid camptothecin bound to phytosphingosine.⁴⁸⁴ This ligand acting as an adjuvant strongly increases the sensitizing capacity of Pru p 3 via CD1-mediated activation of invariant Natural Killer T-cells (iNKTs). Interestingly the structure of Pru p 3 presents significant structural similarities to saposins, small molecules that assist the loading of lipids onto CD1d.⁴⁸⁵ LTP is expressed at two key times of

flower and fruit development in peach, namely, during pollination and during embryo development. Figure 150 shows the crystal structure of Pru p 3 (Table 80). Three IgE-binding epitopes on the LTP molecule have been identified: Pru p 3₁₁-2₀, Pru p 3₃₁-4₀ and Pru p 3₇₁-8₀. These peptides are shared with other fruits including apple, apricot, plum, cherry, orange, strawberry, grape, with a sequence identity ranging from 62 to 81%.¹⁴⁵⁰ Pastorello et al.¹⁴⁵¹ also identified two immunodominant T-cell reactive regions, that have been called Pru p 3₁₂-2₇ and Pru p 3₅₇-7₂. These peptides have the ability to induce the production of IL-4 by Pru p 3-specific T cells after allergen-specific stimulation, reflecting a Th2-dominated response. The stable tertiary conformation of Pru p 3, provides resistance to thermal degradation. *In vitro* IgE-binding ability is maintained after 30 min at 121°C and after 160 min at 100°C. Pru p 3 is as well resistant to proteolytic digestion. After a proteolytic treatment with trypsin, 35% of the molecule remains intact. After proteolysis, three high molecular weight (HMV) peptides and the peptide Pru p 3₅₇-7₂ are released. These peptides have still IgE- and T-binding ability and thus have the potential to either sensitize or induce an allergic reaction¹⁴⁵²

## 2 – The family

Pru p 3 belongs to the family of nonspecific Lipid Transfer Proteins (nsLTP), which includes allergens most frequently involved in food allergic reactions in the adult population from the Mediterranean area.¹⁶⁹⁸ LTP belong to the superfamily of Prolamins including several families like alpha-amylase inhibitors; 2S albumins and nsLTP. All members of the prolamin superfamily share the conserved pattern of eight cysteine residues; nsLTP as a difference to other members of the superfamily are not restricted to seed tissues but ubiquitously expressed throughout the plant.

<figure><img src="image_273.png" /><figcaption>FIGURE 150 Crystal structure of peach Pru p 3, prototypic member of the family of plant nonspecific lipid transfer protein pan-allergens. Protein chains are colored. The figure was generated using PyMOL Molecular Graphics System v1.6.</figcaption></figure>
# C03 – Nonspecific lipid transfer proteins (nsLTP)

Elide Anna Pastorello, Francisca Gomez, Domingo Barber

## Highlights

* Nonspecific lipid transfer proteins (nsLTP) are the most prevalent plant-food allergens in Southern Europe.

* The clinical reactions can be systemic and severe, especially when not associated to birch pollinosis.

* Pru p 3, the major allergen of peach, plays a precursor role in the sensitization to other nsLTP.

* Relevant nsLTP containing plant-foods belong not only to the *Rosaceae* family but also to the nut group and to cereals, such as wheat, maize, and rice.

## 1 – The protein

Pru p 3, the major allergen of peach, was the first nsLTP to be fully identified and characterized as a relevant food allergen. It is the most broadly recognized allergen of the family; however, there are patients sensitized to nsLTP that might not be sensitized to Pru p 3. It is in vascular tissue and in the outer cell layers of the plant organs and it essentially concentrates in the pericarp of fruits, whereas the pulp contains levels around 220-fold lower than peel. Peach fuzz contains large amounts of Pru p 3. It is a small basic protein of 91 amino acids, with a molecular weight of 9,178 Da. It contains a highly conserved domain consisting of alpha-helices that harbor eight cysteine residues, a distinctive sign of belonging to the Prolamin superfamily. The cysteine residues form four intramolecular disulphide bridges, which makes the protein resistant to high temperature and pH changes. The four disulphide bridges are responsible for the LTP compact folding, which forms a tunnel-like hydrophobic cavity running through the whole molecule. The Pru p 3 molecule is very flexible; therefore, the volume of the inner cavity can change significantly, and the protein can bind various lipidic ligands. The changes of the allergen conformation, which are related to ligand binding, alter the protein surface and this can modify its IgE-binding properties. This has been proposed for Pru p 3, where using an in silico approach, only the binding with oleic acid and not with stearic acid conditioned the exposure of the C-terminal loop that is a major IgE-binding epitope thus increasing the IgE-binding properties of this molecule.⁴⁸² Moreover, the natural ligand of Pru p 3 has been recently identified as a derivative of the alkaloid camptothecin bound to phytosphingosine.⁴⁸⁴ This ligand acting as an adjuvant strongly increases the sensitizing capacity of Pru p 3 via CD1-mediated activation of invariant Natural Killer T-cells (iNKTs). Interestingly the structure of Pru p 3 presents significant structural similarities to saposins, small molecules that assist the loading of lipids onto CD1d.⁴⁸⁵ LTP is expressed at two key times of

flower and fruit development in peach, namely, during pollination and during embryo development. Figure 150 shows the crystal structure of Pru p 3 (Table 80). Three IgE-binding epitopes on the LTP molecule have been identified: Pru p 3₁₁-2₀, Pru p 3₃₁-4₀ and Pru p 3₇₁-8₀. These peptides are shared with other fruits including apple, apricot, plum, cherry, orange, strawberry, grape, with a sequence identity ranging from 62 to 81%.¹⁴⁵⁰ Pastorello et al.¹⁴⁵¹ also identified two immunodominant T-cell reactive regions, that have been called Pru p 3₁₂-2₇ and Pru p 3₅₇-7₂. These peptides have the ability to induce the production of IL-4 by Pru p 3-specific T cells after allergen-specific stimulation, reflecting a Th2-dominated response. The stable tertiary conformation of Pru p 3, provides resistance to thermal degradation. *In vitro* IgE-binding ability is maintained after 30 min at 121°C and after 160 min at 100°C. Pru p 3 is as well resistant to proteolytic digestion. After a proteolytic treatment with trypsin, 35% of the molecule remains intact. After proteolysis, three high molecular weight (HMV) peptides and the peptide Pru p 3₅₇-7₂ are released. These peptides have still IgE- and T-binding ability and thus have the potential to either sensitize or induce an allergic reaction¹⁴⁵²

## 2 – The family

Pru p 3 belongs to the family of nonspecific Lipid Transfer Proteins (nsLTP), which includes allergens most frequently involved in food allergic reactions in the adult population from the Mediterranean area.¹⁶⁹⁸ LTP belong to the superfamily of Prolamins including several families like alpha-amylase inhibitors; 2S albumins and nsLTP. All members of the prolamin superfamily share the conserved pattern of eight cysteine residues; nsLTP as a difference to other members of the superfamily are not restricted to seed tissues but ubiquitously expressed throughout the plant.

<figure><img src="image_279.png" /><figcaption>FIGURE 150 Crystal structure of peach Pru p 3, prototypic member of the family of plant nonspecific lipid transfer protein pan-allergens. Protein chains are colored. The figure was generated using PyMOL Molecular Graphics System v1.6.</figcaption></figure>
<figure><img src="image_274.png" /><figcaption>FIGURE 151 Cross-reactivity due to nsLTP molecules between different allergenic sources. Continuous lines indicate a high degree of cross-reactivity among the *Rosaceae* family. Dashed lines indicate partial cross-reactivity.</figcaption></figure>

<table><caption>TABLE 80 Protein characteristics</caption><thead><tr><th colspan="2">Protein characteristics</th></tr></thead><tbody><tr><td>Allergen source</td><td><i>Prunus persica</i>, peach</td></tr><tr><td>Protein family</td><td>Lipid Transfer Proteins</td></tr><tr><td>UniProtKB accession No.</td><td>P81402</td></tr><tr><td>Crystal available</td><td>Yes</td></tr><tr><td>Molecular structure</td><td>Mainly alpha-helix</td></tr><tr><td>Molecular weight</td><td>9,178 Da</td></tr><tr><td>Lenght</td><td>91 amino acids</td></tr><tr><td>Ligand binding</td><td>Mainly hydrophobic ligands</td></tr><tr><td>Dimerization</td><td>No</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>4</td></tr><tr><td>Isoelectric point</td><td>9.25</td></tr><tr><td>Distribution</td><td>Seeds, fruits, leaves, roots, pollens</td></tr></tbody></table>

LTP concentration is variable and depends on maturity, storage conditions, and cultivar of the fruit. Besides nsLTP have a role in the transport of hydrophobic molecules that compose the cutin and suberin layers of plant tissues. It has been suggested that nsLTP could be involved in plant defense against bacterial and fungal pathogens, and therefore they are also classified as Pathogenesis-Related Proteins type 14, (PR-14). Following the line of studies with Pru p 3, it has been shown that natural ligands transported by other allergenic LTP such as those from Triticum aestivum (wheat), Artemisia vulgaris (mugwort), Parietaria judaica (pellitory of the wall) and Olea europea (olive) were similar to those transported by Pru p 3. The authors also

demonstrated that Phytosphingosin could act as a functional analogue of human SPH (sphingosine) being converted by the epithelial enzyme SphK1 into PHS1P, a phosphorylated metabolite analogue of S1P that plays a significant role in the pathogenesis of asthma and allergy.⁴⁸³,¹⁶⁹⁸ To date, the International Union of Immunological Societies Allergen Nomenclature Sub-committee lists 46 allergenic LTP molecules originated from fruits, pollen of trees and weeds, vegetables, nuts, and seeds, as well as latex. (Figure 151) based on their molecular mass, nsLTP are grouped into two types, LTP1 (9–10 kDa; around 90 amino acids) and LTP2 (6-7 kDa; around 70 amino acids). However, the majority of allergenic nsLTP belong to the nsLTP1 type. The most characteristic members are included in (Table 81). LTP are the most important allergens of the *Prunoideae* subfamily such as peach, apricot, plum and cherry. IgE cross-reactivity has been observed within the *Rosaceae* family (high degree) and with citrus fruits, grape, tomato, vegetables (asparagus, lettuce, etc.), nuts (walnut, walnut, peanut, etc.), maize, onion, carrot, rice, and spelt (partial cross-reactivity).⁴⁸⁵ The Arg39/Thr40 epitope is well conserved in *Rosaceae* nsLTPs and only partly in cereal nsLTP (Figure 153). Besides, relevant allergens from Parietaria, Artemisia, Platanus and Olea pollen are also members of the LTP family, but they show rather low (Artemisia and Platanus) or absent (Parietaria and Olea) cross-reactivity with Pru p 3 as a consequence of the lower sequence identity (<35%), and different length.¹⁴⁵¹ (Figure 154) shows conserved and divergent residues between *Rosaceae* nsLTP (A) and sequence identity between Pru p 3 and Art v 3 (B) or Ara h 9 (C). Recently Skypala et al., have reviewed extensively the different aspects of nsLTP structure, cross-reactivity and epidemiology.³⁵
<figure><img src="image_280.png" /><figcaption>FIGURE 151 Cross-reactivity due to nsLTP molecules between different allergenic sources. Continuous lines indicate a high degree of cross-reactivity among the *Rosaceae* family. Dashed lines indicate partial cross-reactivity.</figcaption></figure>

<table><caption>TABLE 80 Protein characteristics</caption><thead><tr><th colspan="2">Protein characteristics</th></tr></thead><tbody><tr><td>Allergen source</td><td><i>Prunus persica</i>, peach</td></tr><tr><td>Protein family</td><td>Lipid Transfer Proteins</td></tr><tr><td>UniProtKB accession No.</td><td>P81402</td></tr><tr><td>Crystal available</td><td>Yes</td></tr><tr><td>Molecular structure</td><td>Mainly alpha-helix</td></tr><tr><td>Molecular weight</td><td>9,178 Da</td></tr><tr><td>Lenght</td><td>91 amino acids</td></tr><tr><td>Ligand binding</td><td>Mainly hydrophobic ligands</td></tr><tr><td>Dimerization</td><td>No</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>4</td></tr><tr><td>Isoelectric point</td><td>9.25</td></tr><tr><td>Distribution</td><td>Seeds, fruits, leaves, roots, pollens</td></tr></tbody></table>

LTP concentration is variable and depends on maturity, storage conditions, and cultivar of the fruit. Besides nsLTP have a role in the transport of hydrophobic molecules that compose the cutin and suberin layers of plant tissues. It has been suggested that nsLTP could be involved in plant defense against bacterial and fungal pathogens, and therefore they are also classified as Pathogenesis-Related Proteins type 14, (PR-14). Following the line of studies with Pru p 3, it has been shown that natural ligands transported by other allergenic LTP such as those from Triticum aestivum (wheat), Artemisia vulgaris (mugwort), Parietaria judaica (pellitory of the wall) and Olea europea (olive) were similar to those transported by Pru p 3. The authors also

demonstrated that Phytosphingosin could act as a functional analogue of human SPH (sphingosine) being converted by the epithelial enzyme SphK1 into PHS1P, a phosphorylated metabolite analogue of S1P that plays a significant role in the pathogenesis of asthma and allergy.⁴⁸³,¹⁶⁹⁸ To date, the International Union of Immunological Societies Allergen Nomenclature Sub-committee lists 46 allergenic LTP molecules originated from fruits, pollen of trees and weeds, vegetables, nuts, and seeds, as well as latex. (Figure 151) based on their molecular mass, nsLTP are grouped into two types, LTP1 (9–10 kDa; around 90 amino acids) and LTP2 (6-7 kDa; around 70 amino acids). However, the majority of allergenic nsLTP belong to the nsLTP1 type. The most characteristic members are included in (Table 81). LTP are the most important allergens of the *Prunoideae* subfamily such as peach, apricot, plum and cherry. IgE cross-reactivity has been observed within the *Rosaceae* family (high degree) and with citrus fruits, grape, tomato, vegetables (asparagus, lettuce, etc.), nuts (walnut, walnut, peanut, etc.), maize, onion, carrot, rice, and spelt (partial cross-reactivity).⁴⁸⁵ The Arg39/Thr40 epitope is well conserved in *Rosaceae* nsLTPs and only partly in cereal nsLTP (Figure 153). Besides, relevant allergens from Parietaria, Artemisia, Platanus and Olea pollen are also members of the LTP family, but they show rather low (Artemisia and Platanus) or absent (Parietaria and Olea) cross-reactivity with Pru p 3 as a consequence of the lower sequence identity (<35%), and different length.¹⁴⁵¹ (Figure 154) shows conserved and divergent residues between *Rosaceae* nsLTP (A) and sequence identity between Pru p 3 and Art v 3 (B) or Ara h 9 (C). Recently Skypala et al., have reviewed extensively the different aspects of nsLTP structure, cross-reactivity and epidemiology.³⁵
<table><caption>TABLE 81 Representative members of the nsLTP family and cross-reactivity between them</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td>Plant foods</td><td></td><td></td></tr><tr><td rowspan="3">Rosaceae</td><td>Peach (Prunus persica)</td><td>Pru p 3</td></tr><tr><td>Apple (Malus domestica)</td><td>Mal d 3</td></tr><tr><td>Cherry (Prunus avium)</td><td>Pru av 3</td></tr><tr><td>Vitaceae</td><td>Grape (Vitis vinifera)</td><td>Vit v 1</td></tr><tr><td>Rutaceae</td><td>Orange (Citrus sinensis)</td><td>Cit s 3</td></tr><tr><td>Solanaceae</td><td>Tomato (Lycopersicon esculentum)</td><td>Lyc e 3</td></tr><tr><td>Corylaceae</td><td>Hazelnut (Corylus avellana)</td><td>Cor a 8</td></tr><tr><td>Juglandaceae</td><td>Walnut (Juglans regia)</td><td>Jug r 3</td></tr><tr><td>Fabaceae</td><td>Peanut (Arachis hypogaea)</td><td>Ara h 9</td></tr><tr><td>Asteraceae</td><td>Lettuce (Lactuca sativa)</td><td>Lac s 1</td></tr><tr><td rowspan="2">Poaceae</td><td>Maize (Zea mays)</td><td>Zea m 14</td></tr><tr><td>Wheat (Triticum aestivum)</td><td>Tri a 14</td></tr><tr><td>Occupational allergens</td><td></td><td></td></tr><tr><td>Euphorbiaceae</td><td>Latex (Hevea brasiliensis)</td><td>Heb b 12</td></tr><tr><td>Asparagaceae</td><td>Asparagus (Asparagus officinalis)</td><td>Aspa o 1</td></tr><tr><td>Pollen</td><td></td><td></td></tr><tr><td rowspan="2">Urticaceae</td><td>Parietaria (Parietaria judaica)</td><td>Par j 1</td></tr><tr><td>Ragweed (Ambrosia artemisiifolia)</td><td>Amb a 6</td></tr><tr><td rowspan="2">Asteraceae</td><td>Mugwort (Artemisia vulgaris)</td><td>Art v 3</td></tr><tr><td>Olive (Olea europaea)</td><td>Ole e 7</td></tr><tr><td>Platanaceae</td><td>Plane (Platanus acerifolia)</td><td>Pla a 3</td></tr></tbody></table>

A complete list of described allergenic LTPs can be found at: http://allergen.org/

## 3 – Clinical relevance

Clinically relevant nsLTP have been described both in foods and pol-lens. The real frequency of LTP sensitization in Europe is unknown, however, in an epidemiological survey performed in Spain<sup>280,585</sup> on pollen allergic patients, Pru p 3 sensitization affected 11% of the adults and 22% of the children tested. These data suggest that ap-proximately 2% of adults and 4% of children show positive sIgE to Pru p 3 (assuming a 20% prevalence of pollen allergy). Approximately 50% of the patients that were sensitized to Pru p 3 referred food allergy. Considering the patients that are not pollen allergic and exclusively LTP-sensitized, the LTP-syndrome represents the most frequent type of food allergy in adults and adolescents in Southern European countries. By contrast, this syndrome shows a low preva-lence in Central and Northern Europe. From a clinical point of view, the LTP-syndrome presents some peculiar aspects, which need to be known for the appropriate management of LTP allergic patients.

### 3.1. Clinical reactivity

A remarkable feature of LTP sensitization is the high variability of its clinical presentation, ranging from mild contact urticaria to ana-phylaxis.<sup>1454,1455,1699</sup> Different studies described the robust associ-ation between Pru p 3 positivity and the severity of systemic allergic symptoms. Even recently, in a component-resolved diagnosis based (CRD) study conducted on an adult cohort of 54 patients with a his-tory of plant-food allergen-induced anaphylaxis, the authors found

that nsLTP were one of the most frequent causes of anaphylaxis.<sup>1455</sup> In most of the cases systemic symptoms are preceded by oral allergy syndrome (OAS) manifestations, probably because the route of ex-posure is the oral mucosa. The severity of the reaction seems to be higher when patients are mono-sensitized to LTP and milder when patients are also sensitized to profilin or PR-10.<sup>1128</sup> level of specific IgE to Pru p 3 does not correlate with the severity of reactions. Patients can present restricted IgE recognition to one LTP or a broad recognition spectrum (LTP syndrome). Moreover, the most severe reactions are frequently associated to different co-factors such as NSAIDs intake or exercise.<sup>1454</sup>

### 3.2. Geographical differences

Another peculiar aspect of the LTP-syndrome stems from the clear-cut geographical difference in sensitization. The severe LTP-induced clinical manifestations seen in Mediterranean populations are usually in contrast to mild clinical manifestations of the oral allergy syndrome that are associated to Birch pollen allergy caused by Bet v 1 and Bet v 2 sensitization in Central and Northern Europe. In a peach allergy model, patients were prone to suffer severe reactions in areas with low level of Fagales pollen, usually non pollen allergics, while pollen co-sensitized subjects presented milder symptoms induced by Pru p 3. This finding also has been confirmed by other Authors in an apple model.<sup>1449</sup> In a European collaborative study on apple al-lergy, Mal d 3 sensitization was significantly more frequent in Spain, an almost birch-free area, as compared to birch-rich countries such
<table><caption>TABLE 81 Representative members of the nsLTP family and cross-reactivity between them</caption><thead><tr><th>Botanical family</th><th>Allergen source</th><th>Allergen</th></tr></thead><tbody><tr><td>Plant foods</td><td></td><td></td></tr><tr><td rowspan="3">Rosaceae</td><td>Peach (Prunus persica)</td><td>Pru p 3</td></tr><tr><td>Apple (Malus domestica)</td><td>Mal d 3</td></tr><tr><td>Cherry (Prunus avium)</td><td>Pru av 3</td></tr><tr><td>Vitaceae</td><td>Grape (Vitis vinifera)</td><td>Vit v 1</td></tr><tr><td>Rutaceae</td><td>Orange (Citrus sinensis)</td><td>Cit s 3</td></tr><tr><td>Solanaceae</td><td>Tomato (Lycopersicon esculentum)</td><td>Lyc e 3</td></tr><tr><td>Corylaceae</td><td>Hazelnut (Corylus avellana)</td><td>Cor a 8</td></tr><tr><td>Juglandaceae</td><td>Walnut (Juglans regia)</td><td>Jug r 3</td></tr><tr><td>Fabaceae</td><td>Peanut (Arachis hypogaea)</td><td>Ara h 9</td></tr><tr><td>Asteraceae</td><td>Lettuce (Lactuca sativa)</td><td>Lac s 1</td></tr><tr><td rowspan="2">Poaceae</td><td>Maize (Zea mays)</td><td>Zea m 14</td></tr><tr><td>Wheat (Triticum aestivum)</td><td>Tri a 14</td></tr><tr><td>Occupational allergens</td><td></td><td></td></tr><tr><td>Euphorbiaceae</td><td>Latex (Hevea brasiliensis)</td><td>Heb b 12</td></tr><tr><td>Asparagaceae</td><td>Asparagus (Asparagus officinalis)</td><td>Aspa o 1</td></tr><tr><td>Pollen</td><td></td><td></td></tr><tr><td rowspan="2">Urticaceae</td><td>Parietaria (Parietaria judaica)</td><td>Par j 1</td></tr><tr><td>Ragweed (Ambrosia artemisiifolia)</td><td>Amb a 6</td></tr><tr><td rowspan="2">Asteraceae</td><td>Mugwort (Artemisia vulgaris)</td><td>Art v 3</td></tr><tr><td>Olive (Olea europaea)</td><td>Ole e 7</td></tr><tr><td>Platanaceae</td><td>Plane (Platanus acerifolia)</td><td>Pla a 3</td></tr></tbody></table>

A complete list of described allergenic LTPs can be found at: http://allergen.org/

## 3 – Clinical relevance

Clinically relevant nsLTP have been described both in foods and pol- lens. The real frequency of LTP sensitization in Europe is unknown, however, in an epidemiological survey performed in Spain<sup>280,585</sup> on pollen allergic patients, Pru p 3 sensitization affected 11% of the adults and 22% of the children tested. These data suggest that ap- proximately 2% of adults and 4% of children show positive sIgE to Pru p 3 (assuming a 20% prevalence of pollen allergy). Approximately 50% of the patients that were sensitized to Pru p 3 referred food allergy. Considering the patients that are not pollen allergic and exclusively LTP-sensitized, the LTP-syndrome represents the most frequent type of food allergy in adults and adolescents in Southern European countries. By contrast, this syndrome shows a low preva- lence in Central and Northern Europe. From a clinical point of view, the LTP-syndrome presents some peculiar aspects, which need to be known for the appropriate management of LTP allergic patients.

### 3.1. Clinical reactivity

A remarkable feature of LTP sensitization is the high variability of its clinical presentation, ranging from mild contact urticaria to ana- phylaxis.<sup>1454,1455,1699</sup> Different studies described the robust associ- ation between Pru p 3 positivity and the severity of systemic allergic symptoms. Even recently, in a component-resolved diagnosis based (CRD) study conducted on an adult cohort of 54 patients with a his- tory of plant-food allergen-induced anaphylaxis, the authors found

that nsLTP were one of the most frequent causes of anaphylaxis.<sup>1455</sup> In most of the cases systemic symptoms are preceded by oral allergy syndrome (OAS) manifestations, probably because the route of ex- posure is the oral mucosa. The severity of the reaction seems to be higher when patients are mono-sensitized to LTP and milder when patients are also sensitized to profilin or PR-10.<sup>1128</sup> level of specific IgE to Pru p 3 does not correlate with the severity of reactions. Patients can present restricted IgE recognition to one LTP or a broad recognition spectrum (LTP syndrome). Moreover, the most severe reactions are frequently associated to different co-factors such as NSAIDs intake or exercise.<sup>1454</sup>

### 3.2. Geographical differences

Another peculiar aspect of the LTP-syndrome stems from the clear-cut geographical difference in sensitization. The severe LTP- induced clinical manifestations seen in Mediterranean populations are usually in contrast to mild clinical manifestations of the oral allergy syndrome that are associated to Birch pollen allergy caused by Bet v 1 and Bet v 2 sensitization in Central and Northern Europe. In a peach allergy model, patients were prone to suffer severe reactions in areas with low level of Fagales pollen, usually non pollen allergics, while pollen co-sensitized subjects presented milder symptoms induced by Pru p 3. This finding also has been confirmed by other Authors in an apple model.<sup>1449</sup> In a European collaborative study on apple al- lergy, Mal d 3 sensitization was significantly more frequent in Spain, an almost birch-free area, as compared to birch-rich countries such
as Netherland, Austria and northern Italy.³⁵,¹⁴⁴⁹ These correlations suggest that Birch pollen exposure, typical of Central and Northern European populations, may confer a sort of immune protection to LTP sensitization. Recently the role of LTP as food and pollen allergens even outside the Mediterranean area has been reviewed and demonstrated to be expressed in different phenotypes depending upon the sources and the intensity of exposure.³⁵ In general the sensitization pattern to LTP outside the Mediterranean area differs from that of Southern Europe, with an overall lower prevalence and an apparent association to pollen cross-reactive LTP.³⁵,¹⁴⁴⁹ A typical example is the cross-reactivity between Pru p 3 and Art v 3 in China.¹²⁵⁵

### 3.3. Pediatric patients

Children sensitized to Pru p 3 present clinical symptoms earlier than those sensitized to other pollen related allergens.¹¹⁴⁸ In a clinical study carried out in Italy in adults, a total of 26 out of 48 subjects that were Pru p 3 positives alone, presented peach-related allergy symptoms between 2-15 years, earlier than those sensitized only to Pru p 1 and/or Pru p 4 allergens. Furthermore, in the same patients, Pru p 3 sensitization correlated with the development of allergic reactions to a higher number of plant-foods than Pru p 1 and Pru p 4 sensitization alone.

### 3.4. Role of Pru p 3

Peach is the most frequent cause for nsLTP allergy, and Pru p 3-sensitization seems to play a precursor role in the sensitization to other nsLTP. The most frequently involved plant-foods are fruits of the *Rosaceae* family such as apple, plum, apricot, cherry and pear. However, there are also botanically unrelated LTP-containing plant foods that appear to be strongly associated with peach, particularly, in the nut group: walnut, hazelnut and peanut have been described as foods eliciting not only OAS but also severe systemic reactions in LTP-sensitized subjects. Also, the most important cereals such as wheat, maize and rice can cause systemic reactions of various grades of severity as confirmed by double-blind placebo-controlled food challenge (DBPCFC) in LTP-allergic patients. In particular wheat has been recently described as cause of exercise related anaphylaxis in three young LTP-sensitized patients. LTP allergens also have been identified in green bean, fennel, orange, kiwi and lentil when using the sera of allergic patients previously sensitized to peach LTP. A large number of the aforementioned studies have indeed demonstrated that Pru p 3-sensitization dominates the immune response to LTP in the other foods and that peach is almost always the food initiating the LTP allergy syndrome. However, clinical reactivity is not invariably a direct consequence of the cross-reactivity.³⁵,¹⁴⁴⁹ In many cases LTP-containing, Pru p 3 cross-reactive foods can be tolerated. Recently, an alternative sensitization route has been reported, independent from Pru p 3, connected to marijuana inhalation mediated by Can s 3, the nsLTP from Cannabis sativa.³⁵

### 3.5. nsLTP and respiratory allergens

As shown in Table 81, nsLTP have been identified as major and minor allergens in different tree and weed pollens. It is worth to

pointout that frequently airborne sensitization to pollen LTPs is associated with more severe clinical phenotypes. Parietaria pollen is the only pollen with a nsLTP as the major allergen. Accepted threshold pollen levels for sensitization are low and clinically, Parietaria pollinosis is often linked to asthma. Ole e 7, the nsLTP from olive pollen, has been reported to identify a severe allergic olive pollen phenotype, with increased risk of asthma and side-reactions during immunotherapy. In areas of heavy olive pollen exposure this allergen can sensitize up to 50% of pollen-allergic population.⁵⁸⁵ It has been recently reported that Ole e 7 could play a new role as primary sensitizer in these areas, leading to peach nsLTP sensitization. This co-sensitization process would occur because of the cross-reactivity between Ole e 7 and Pru p 3 observed in some allergic patients.¹⁴⁵⁶ Art v 3 and Pla a 3, are minor pollen allergens and display partial cross-reactivity with Pru p 3 (Figure 152). As a consequence, sIgE to either *Artemisia* or *Platanus* should always be considered for potential cross-reactivity and should be assessed in connection with major pollen allergens and Pru p 3. In areas with high *Platanus* or *Artemisia* exposure, sensitization to these pollen LTP has been associated with a more complex recognition pattern in nsLTP food allergic patients.¹⁴⁵⁷ It has also been reported that Pru p 3 is able to induce respiratory symptoms in areas with extensive orchard tree cultivation.³⁵ Further, asparagus nsLTP has been described as an occupational allergen able to induce respiratory symptoms.¹⁴⁵⁸ As previously mentioned, Can s 3, the nsLTP from Cannabis can induce respiratory allergy.³⁵

## 4 – Clinical management

### 4.1. Identification of clinically relevant allergens

The clinical history should be aimed first at identifying reactions to plant-foods most frequently involved in typical reactions of the LTP syndrome (Figure 153). Primarily peach, apple and other *Rosaceae* fruits and second a group of seeds that frequently are linked to LTP allergy as walnut, hazelnut, peanut, maize, wheat rice, or other beans. However, it should be taken into account that LTP are present in all vegetable tissues and can always be etiological proteins in vegetable-mediated allergies. In spite of this, the presence of nLTP in a plant-food is not a reason to avoid it if tolerated until that moment as the contact with the intestinal immune system may help in maintaining tolerance. A list containing the nsLTP-rich foods should be avoided and the situation for each single food should be evaluated before its exclusion.

### 4.2. Role of cofactors

A second critical point of the diagnosis is the consideration that severe reactions might be linked to cofactors such as exercise, alcohol intake, anti-acids and NSAIDs and thus LTP potential involvement should also always be evaluated associated to those.¹⁴⁵⁴ A third point to consider is that many times severe LTP-associated clinical reactions take place in patients that are not pollen allergic.
as Netherland, Austria and northern Italy.³⁵,¹⁴⁴⁹ These correlations suggest that Birch pollen exposure, typical of Central and Northern European populations, may confer a sort of immune protection to LTP sensitization. Recently the role of LTP as food and pollen allergens even outside the Mediterranean area has been reviewed and demonstrated to be expressed in different phenotypes depending upon the sources and the intensity of exposure.³⁵ In general the sensitization pattern to LTP outside the Mediterranean area differs from that of Southern Europe, with an overall lower prevalence and an apparent association to pollen cross-reactive LTP.³⁵,¹⁴⁴⁹ A typical example is the cross-reactivity between Pru p 3 and Art v 3 in China.¹²⁵⁵

### 3.3. Pediatric patients

Children sensitized to Pru p 3 present clinical symptoms earlier than those sensitized to other pollen related allergens.¹¹⁴⁸ In a clinical study carried out in Italy in adults, a total of 26 out of 48 subjects that were Pru p 3 positives alone, presented peach-related allergy symptoms between 2-15 years, earlier than those sensitized only to Pru p 1 and/or Pru p 4 allergens. Furthermore, in the same patients, Pru p 3 sensitization correlated with the development of allergic reactions to a higher number of plant-foods than Pru p 1 and Pru p 4 sensitization alone.

### 3.4. Role of Pru p 3

Peach is the most frequent cause for nsLTP allergy, and Pru p 3-sensitization seems to play a precursor role in the sensitization to other nsLTP. The most frequently involved plant-foods are fruits of the *Rosaceae* family such as apple, plum, apricot, cherry and pear. However, there are also botanically unrelated LTP-containing plant foods that appear to be strongly associated with peach, particularly, in the nut group: walnut, hazelnut and peanut have been described as foods eliciting not only OAS but also severe systemic reactions in LTP-sensitized subjects. Also, the most important cereals such as wheat, maize and rice can cause systemic reactions of various grades of severity as confirmed by double-blind placebo-controlled food challenge (DBPCFC) in LTP-allergic patients. In particular wheat has been recently described as cause of exercise related anaphylaxis in three young LTP-sensitized patients. LTP allergens also have been identified in green bean, fennel, orange, kiwi and lentil when using the sera of allergic patients previously sensitized to peach LTP. A large number of the aforementioned studies have indeed demonstrated that Pru p 3-sensitization dominates the immune response to LTP in the other foods and that peach is almost always the food initiating the LTP allergy syndrome. However, clinical reactivity is not invariably a direct consequence of the cross-reactivity.³⁵,¹⁴⁴⁹ In many cases LTP-containing, Pru p 3 cross-reactive foods can be tolerated. Recently, an alternative sensitization route has been reported, independent from Pru p 3, connected to marijuana inhalation mediated by Can s 3, the nsLTP from Cannabis sativa.³⁵

### 3.5. nsLTP and respiratory allergens

As shown in Table 81, nsLTP have been identified as major and minor allergens in different tree and weed pollens. It is worth to

pointout that frequently airborne sensitization to pollen LTPs is associated with more severe clinical phenotypes. Parietaria pollen is the only pollen with a nsLTP as the major allergen. Accepted threshold pollen levels for sensitization are low and clinically, Parietaria pollinosis is often linked to asthma. Ole e 7, the nsLTP from olive pollen, has been reported to identify a severe allergic olive pollen phenotype, with increased risk of asthma and side-reactions during immunotherapy. In areas of heavy olive pollen exposure this allergen can sensitize up to 50% of pollen-allergic population.⁵⁸⁵ It has been recently reported that Ole e 7 could play a new role as primary sensitizer in these areas, leading to peach nsLTP sensitization. This co-sensitization process would occur because of the cross-reactivity between Ole e 7 and Pru p 3 observed in some allergic patients.¹⁴⁵⁶ Art v 3 and Pla a 3, are minor pollen allergens and display partial cross-reactivity with Pru p 3 (Figure 152). As a consequence, sIgE to either *Artemisia* or *Platanus* should always be considered for potential cross-reactivity and should be assessed in connection with major pollen allergens and Pru p 3. In areas with high *Platanus* or *Artemisia* exposure, sensitization to these pollen LTP has been associated with a more complex recognition pattern in nsLTP food allergic patients.¹⁴⁵⁷ It has also been reported that Pru p 3 is able to induce respiratory symptoms in areas with extensive orchard tree cultivation.³⁵ Further, asparagus nsLTP has been described as an occupational allergen able to induce respiratory symptoms.¹⁴⁵⁸ As previously mentioned, Can s 3, the nsLTP from Cannabis can induce respiratory allergy.³⁵

## 4 – Clinical management

### 4.1. Identification of clinically relevant allergens

The clinical history should be aimed first at identifying reactions to plant-foods most frequently involved in typical reactions of the LTP syndrome (Figure 153). Primarily peach, apple and other *Rosaceae* fruits and second a group of seeds that frequently are linked to LTP allergy as walnut, hazelnut, peanut, maize, wheat rice, or other beans. However, it should be taken into account that LTP are present in all vegetable tissues and can always be etiological proteins in vegetable-mediated allergies. In spite of this, the presence of nLTP in a plant-food is not a reason to avoid it if tolerated until that moment as the contact with the intestinal immune system may help in maintaining tolerance. A list containing the nsLTP-rich foods should be avoided and the situation for each single food should be evaluated before its exclusion.

### 4.2. Role of cofactors

A second critical point of the diagnosis is the consideration that severe reactions might be linked to cofactors such as exercise, alcohol intake, anti-acids and NSAIDs and thus LTP potential involvement should also always be evaluated associated to those.¹⁴⁵⁴ A third point to consider is that many times severe LTP-associated clinical reactions take place in patients that are not pollen allergic.
<figure><img src="image_275.png" /><figcaption>FIGURE 152 Homology between members of the nsLTP family. (A) Surface residues on Pru p 3 conserved among 8 *Rosaceae* fruit nsLTP proteins. Red: residues conserved in 90% or more proteins. Orange: residues conserved in 40-90% of the proteins (gradient); White: residues conserved in less than 40% of the proteins. (B) Surface residues conserved among Pru p 3 and 4 isoforms of Art v 3. Red: residues conserved in 90% or more Art v 3 isoforms. Orange: residues conserved in 40-90% Art v 3 isoform (gradient). White: residues conserved in less than 40% Art v 3 isoform. (C) Surface residues conserved among Pru p 3 and Ara h 9. Red: residues conserved in Pru p 3 and Ara h 9. White: no conserved residues</figcaption></figure>

<figure><img src="image_276.png" /><figcaption>FIGURE 153 Diagnostic algorithms using molecular IgE diagnostics in the case of positive SPT/extract-based IgE test to (A) peanut; (B) peach; (C) hazelnut or (D) apple. LTP allergens are displayed in bold.</figcaption></figure>
<figure><img src="image_281.png" /><figcaption>FIGURE 152 Homology between members of the nsLTP family. (A) Surface residues on Pru p 3 conserved among 8 *Rosaceae* fruit nsLTP proteins. Red: residues conserved in 90% or more proteins. Orange: residues conserved in 40-90% of the proteins (gradient); White: residues conserved in less than 40% of the proteins. (B) Surface residues conserved among Pru p 3 and 4 isoforms of Art v 3. Red: residues conserved in 90% or more Art v 3 isoforms. Orange: residues conserved in 40-90% Art v 3 isoform (gradient). White: residues conserved in less than 40% Art v 3 isoform. (C) Surface residues conserved among Pru p 3 and Ara h 9. Red: residues conserved in Pru p 3 and Ara h 9. White: no conserved residues</figcaption></figure>

<figure><img src="image_282.png" /><figcaption>FIGURE 153 Diagnostic algorithms using molecular IgE diagnostics in the case of positive SPT/extract-based IgE test to (A) peanut; (B) peach; (C) hazelnut or (D) apple. LTP allergens are displayed in bold.</figcaption></figure>
### 4.3. Component-resolved diagnosis (CRD)

Positive skin tests and/or extract specific IgE confirming plant-food sensitization should be further investigated by CRD. Skin prick tests with commercial extracts, and prick-to-prick with fresh vegetable foods should be performed according to European guidelines. Prick-to-prick testing with fresh fruits and vegetables has proven to be more sensitive than SPT with commercial extracts in confirming a history of food allergy to plant-foods. In order to test for LTP sensitization, nsLTP enriched allergenic commercial extracts of apple and peach are now available on the market. They can be useful for the identification of a large number of LTP sensitized patients, as Pru p 3 has proven to be pivotal in LTP syndrome. However, Pru p 3 cannot be considered a general marker of clinical LTP-allergy. As previously mentioned, it shows very limited sequence homology with pollen LTP, such as Par j 1 or Ole e 7, which implicates a very low risk of cross-reactivity. Other LTP such as Tri a 14 connected to baker's asthma, has proven to be an independent sensitizer as well.¹⁴⁵⁹

The key-role of nsLTP as a marker of true food allergy with a high risk of severe systemic reactions, as compared to food allergy (i.e., OAS) due to Bet v 1 and Bet v 2 homologues in birch allergic patients, has prompted the development of assays for detecting anti-LTP specific IgE (ImmunoCAP Specific IgE test) in the diagnosis of plant-foods allergy.

In peach, the simultaneous presence of both, anti-Pru p3 and anti-Pru p1 IgE antibodies seems to lower the risk of severe food allergic reactions.¹⁴⁶⁰ This observation has been confirmed by Ruano Zaragoza et al.¹⁴⁶¹ who found in a population of 431 Pru p 3-sensitized patients that Pru p 1 or Pru p 12 positivity was negatively associated with development of anaphylaxis. Similarly, Mota I et al.¹⁴⁶² found in a group of 43 LTP sensitized patients that the co-sensitization to plant food allergens belonging to PR-10 family could be a preventing factor able to reduce the severity of LTP allergy. In an analysis of severity biomarkers, Bogas G et al. found that profilin positive patients had statistically fewer anaphylactic events compared to patients sensitized exclusively to LTP.¹¹²³ All these data clearly demonstrate the important role of CRD in the diagnosis of LTP syndrome and in phenotyping with regard to severity. The identification of IgE cut-off values correlating with symptom severity could highly increase the efficacy of diagnosis. At present, nine different nsLTPs are available in ImmunoCAP ISAC and fifteen in MADX from Macro Array Diagnostics. The clinical relevance of testing these panels to predict the clinical reactivity pattern is still subject of active research. As a difference to other pan-allergen allergies, it would be necessary to incorporate a broader nsLTP panel for making a correct patient diagnosis. In this context the broad panel offered in MADX provides a new tool to investigate nsLTP allergy. Pru p 3 seems to play a central role in nsLTP-mediated reactions. *In vivo* and *in vitro* diagnostic tools for Pru p 3 are commercially available and their inclusion in general patient screening is advisable, especially in geographic areas where the prevalence of nsLTP sensitization is considerable.

### 4.4. DBPCFCs for LTP-containing plant-foods

The double-blind placebo-controlled food challenge (DBPCFC) is the diagnostic gold standard in food allergy. In routine clinical practice, if multiple challenge tests are not feasible, DBPCFCs for LTP-containing plant-foods should be performed for the most nutritionally relevant or widely consumed foods to minimize unnecessary exclusion from the diet. In some cases, the challenge test should also be performed after exercise given that LTP have been described as foods involved in food-dependent exercise-induced anaphylaxis (FDEIA).¹⁴⁶³

### 4.5. Diagnostic algorithms

Many patients show a progressive clinical recognition of LTP. In some cases, they might be obliged to avoid almost any vegetable. The complexity and intensity of IgE repertoires to the panel of LTP might predict this evolution and is being today a subject of active research.

### 4.6. Treatment

The possibility of desensitization has been explored.⁴⁰¹ In an open controlled study,¹¹⁸¹ 90% of treated patients with a sub-lingual peach extract vaccine, tolerated a whole peach after one year of treatment. Before, about 50% of the patients included had anaphylactic reactions, suggesting that immunotherapy might be a feasible option for the treatment of severe LTP allergy. Moreover, evaluating the effect on concomitant allergy mediated by peanut LTP (Ara h 9), the authors reported a significant clinical benefit, supporting that Pru p 3 can be used for treatment of LTP syndrome. The same authors analysed immunological changes induced by the therapy supporting an induction of a regulatory response to both peach and peanut. A diagnostic algorithm and decision tree for allergen immunotherapy using CRD in nsLTP-mediated allergies has been recently proposed.¹⁴⁶⁴ Unfortunately, this therapy is only available in a limited number of countries. The prescription of adrenaline autoinjectors in cases of previous reactions and in severe risk patients is recommended. An extensive review on the diagnosis and clinical management of LTP allergy has been recently published.¹⁴⁵⁴

## 5 – Clinical cases

### Case 1 (published)¹⁷⁰⁰

Male, 17 years with a 12-year history of peach induced systemic symptoms and sporadic anaphylaxis of unknown origin. Specific IgE were positive for rPru p 3 (34.7 kU/L), while moderate for wheat (1.56 kU/L), maize (4.92 kU/L) and rice (1.46 kU/L). sIgE to omega 5 gliadine was negative. IgE immunoblotting was positive for LTP in the three cereals. Wheat Open Exercise Food Challenge (OEFC) gave rise to an anaphylactic reaction treated with Epinephrine. Rice and maize OEFC were negative. Wheat -free diet allowed the patient to perform agonistic physical activity without any symptoms. He did
### 4.3. Component-resolved diagnosis (CRD)

Positive skin tests and/or extract specific IgE confirming plant-food sensitization should be further investigated by CRD. Skin prick tests with commercial extracts, and prick-to-prick with fresh vegetable foods should be performed according to European guidelines. Prick-to-prick testing with fresh fruits and vegetables has proven to be more sensitive than SPT with commercial extracts in confirming a history of food allergy to plant-foods. In order to test for LTP sensitization, nsLTP enriched allergenic commercial extracts of apple and peach are now available on the market. They can be useful for the identification of a large number of LTP sensitized patients, as Pru p 3 has proven to be pivotal in LTP syndrome. However, Pru p 3 cannot be considered a general marker of clinical LTP-allergy. As previously mentioned, it shows very limited sequence homology with pollen LTP, such as Par j 1 or Ole e 7, which implicates a very low risk of cross-reactivity. Other LTP such as Tri a 14 connected to baker's asthma, has proven to be an independent sensitizer as well.¹⁴⁵⁹

The key-role of nsLTP as a marker of true food allergy with a high risk of severe systemic reactions, as compared to food allergy (i.e., OAS) due to Bet v 1 and Bet v 2 homologues in birch allergic patients, has prompted the development of assays for detecting anti-LTP specific IgE (ImmunoCAP Specific IgE test) in the diagnosis of plant-foods allergy.

In peach, the simultaneous presence of both, anti-Pru p3 and anti-Pru p1 IgE antibodies seems to lower the risk of severe food allergic reactions.¹⁴⁶⁰ This observation has been confirmed by Ruano Zaragoza et al.¹⁴⁶¹ who found in a population of 431 Pru p 3-sensitized patients that Pru p 1 or Phl p 12 positivity was negatively associated with development of anaphylaxis. Similarly, Mota I et al.¹⁴⁶² found in a group of 43 LTP sensitized patients that the co-sensitization to plant food allergens belonging to PR-10 family could be a preventing factor able to reduce the severity of LTP allergy. In an analysis of severity biomarkers, Bogas G et al. found that profilin positive patients had statistically fewer anaphylactic events compared to patients sensitized exclusively to LTP.¹¹²³ All these data clearly demonstrate the important role of CRD in the diagnosis of LTP syndrome and in phenotyping with regard to severity. The identification of IgE cut-off values correlating with symptom severity could highly increase the efficacy of diagnosis. At present, nine different nsLTPs are available in ImmunoCAP ISAC and fifteen in MADX from Macro Array Diagnostics. The clinical relevance of testing these panels to predict the clinical reactivity pattern is still subject of active research. As a difference to other pan-allergen allergies, it would be necessary to incorporate a broader nsLTP panel for making a correct patient diagnosis. In this context the broad panel offered in MADX provides a new tool to investigate nsLTP allergy. Pru p 3 seems to play a central role in nsLTP-mediated reactions. *In vivo* and *in vitro* diagnostic tools for Pru p 3 are commercially available and their inclusion in general patient screening is advisable, especially in geographic areas where the prevalence of nsLTP sensitization is considerable.

### 4.4. DBPCFCs for LTP-containing plant-foods

The double-blind placebo-controlled food challenge (DBPCFC) is the diagnostic gold standard in food allergy. In routine clinical practice, if multiple challenge tests are not feasible, DBPCFCs for LTP-containing plant-foods should be performed for the most nutritionally relevant or widely consumed foods to minimize unnecessary exclusion from the diet. In some cases, the challenge test should also be performed after exercise given that LTP have been described as foods involved in food-dependent exercise-induced anaphylaxis (FDEIA).¹⁴⁶³

### 4.5. Diagnostic algorithms

Many patients show a progressive clinical recognition of LTP. In some cases, they might be obliged to avoid almost any vegetable. The complexity and intensity of IgE repertoires to the panel of LTP might predict this evolution and is being today a subject of active research.

### 4.6. Treatment

The possibility of desensitization has been explored.⁴⁰¹ In an open controlled study,¹¹⁸¹ 90% of treated patients with a sub-lingual peach extract vaccine, tolerated a whole peach after one year of treatment. Before, about 50% of the patients included had anaphylactic reactions, suggesting that immunotherapy might be a feasible option for the treatment of severe LTP allergy. Moreover, evaluating the effect on concomitant allergy mediated by peanut LTP (Ara h 9), the authors reported a significant clinical benefit, supporting that Pru p 3 can be used for treatment of LTP syndrome. The same authors analysed immunological changes induced by the therapy supporting an induction of a regulatory response to both peach and peanut. A diagnostic algorithm and decision tree for allergen immunotherapy using CRD in nsLTP-mediated allergies has been recently proposed.¹⁴⁶⁴ Unfortunately, this therapy is only available in a limited number of countries. The prescription of adrenaline autoinjectors in cases of previous reactions and in severe risk patients is recommended. An extensive review on the diagnosis and clinical management of LTP allergy has been recently published.¹⁴⁵⁴

## 5 – Clinical cases

### Case 1 (published)¹⁷⁰⁰

Male, 17 years with a 12-year history of peach induced systemic symptoms and sporadic anaphylaxis of unknown origin. Specific IgE were positive for rPru p 3 (34.7 kU/L), while moderate for wheat (1.56 kU/L), maize (4.92 kU/L) and rice (1.46 kU/L). slgE to omega 5 gliadine was negative. IgE immunoblotting was positive for LTP in the three cereals. Wheat Open Exercise Food Challenge (OEFC) gave rise to an anaphylactic reaction treated with Epinephrine. Rice and maize OEFC were negative. Wheat -free diet allowed the patient to perform agonistic physical activity without any symptoms. He did
not need to eliminate rice and maize from his diet. Thus, LTP sensitization to rice and maize was due to cross-reactivity with Pru p3. The message is that the challenge is advisable to avoid unnecessary food elimination.

### Case 2 (unpublished real case)

Female, 52-years old with a 32-year history of peach-peel induced contact urticaria and immediate abdominal pain after peach-juice ingestion. She avoided this fruit since the reaction. The patient tolerated other fruits, vegetables, and tree-nuts. After some time, the patient developed a systemic reaction after eating a complete unpeeled apple (generalized urticaria and angioedema). Soon after she referred oral allergy syndrome with walnut, hazelnut and almond and she avoided other nuts as peanuts for fear of having a new reaction.

The patient had a clinical history of allergic rhinitis to olive pollen since she was 15 years old. SPT to aeroallergens was positive for olive, mugwort, plane-tree pollen and HDM. SPT to plant-food was positive to peach peel, apple, hazelnut, almond and peanut. Specific IgE were positive for Pru p 3 (21.50 kU/L), for walnut (21 kU/L), hazelnut (10.20 kU/L) and peanut Ara h 9 (11.5 kU/L); and negative for peanut Ara h 2. We carried-out a peeled apple (154 g) open food challenge and the patient presented lips angioedema after 70 g of the total dose. She was treated with oral loratadine, and the reaction was resolved.

The patient participated in the clinical study with Pru p 3 SLIT with a commercialized peach extract enriched in Pru p 3 (50 µg/mL) (ALK-Abello S.A.). At the inclusion visit, we performed a DBPCFC with unpeeled peach, (300 mL that is a total dose of 2.5 mg of Pru p 3) and peanut (15 units) The patient presented lips angioedema and OAS after 50 mL of peach juice and oral allergy symptoms with VAS of seven with three consecutive dose of peanut (total of seven fried peanut nuts). The patient was included on a clinical trial and was treated during a year with Pru p 3 SLIT, receiving a 10µg daily dose of Pru p 3. After 12 months of treatment, we observed a mild decrease in the size of papule for peach (10 mm area before Pru p 3-SLIT to 6 mm area after Pru p 3-SLIT) but not to peanut. We observed a decrease in slgE to Pru p 3 (12.3 kU/L) and peanut (6.10 kU/L). During the DBPCFC with peach, the patient tolerated the maximum amount of juice and peanut (15 units).

We recommended to include peach and peanut in a free diet (taking the maximum dose tolerated during DBPCFC). This is a clinical case of a patient suffering from LTP-Syndrome, in which SLIT-Pru p 3 has provided an improvement in tolerance not only for peach but also for other foods related to LTP sensitization such as peanuts. After the study the patient continued with the SLIT for two years and we performed OFC with the other nuts (hazelnut and almond) and unpeel-apple, that were introduced in the diet. The patient refused the oral food challenge with walnut.

### Case 3 (unpublished real case)

Male, 30-year with a 24-years old history of peach-peel induced contact urticaria. He avoided this fruit since the reaction. The patient tolerated other fruits, vegetables, and tree-nuts. Ten years after first symptoms, the patient developed a systemic reaction after ingestion of walnut, hazelnut, and peanut (hives, angioedema, shortness of breath, dizziness). Since the reaction with these nuts, the patient avoided all types of tree-nuts, including pistachio and cashew. SPT to aeroallergens was positive to mugwort and plane-tree pollen. SPT to plant-food was positive to peach peel, hazelnut, and peanut; and negative to almond, pistachio, and cashew. Specific IgE were positive for Pru p 3 (20.6 kU/L), moderate for walnut (4.93 kU/L), hazelnut (0.54 kU/L) and peanut Ara h 9 (15 kU/L); and negative for peanut Ara h 2. Almond Open Food Challenge was positive with a total dose of 5 units of fried almond, presenting the patients oral allergy syndrome and lip angioedema that was treated with IM dexchlorpheniramine. Pistachio and cashew OFCs were negative. Upon DBPCFC with unpeeled-peach and peanut, the patient presented OAS and abdominal pain at intermediate peach dose as well as lips angioedema, erythema, and pruritus after 5 units of fried peanut. The patient was included on clinical study and was treated during a year with Pru p 3-SLIT (as in case 2). After 12 months of treatment, we did not observe a decrease in the peach or peanut wheal size, but we observed a decrease in the levels of slgE to Pru p 3 (10.5 kU/L); and peanut (4.3 kU/L). During the DBPCFC with peach the patient tolerated all the amount of juice and peanut (15 units). This is a clinical case of a patient that developed an LTP syndrome, in which Pru p 3-SLIT induced an improvement in tolerance not only for peach but also of other foods related to LTP sensitization such as peanuts. Currently the patient tolerates other nuts as walnut and hazelnut.

## 6 – Tools

CRD by SPT is possible by using peach extracts highly enriched on Pru p 3, (with very low content of other allergens).<sup>1453</sup> As most of the patients with nsLTP-mediated food allergic reactions will be positive to Pru p 3, this extract should be used in a general screening for inhaled as well as food allergy. Complex nsLTP syndrome patients will eventually react to multiple members of the family. Today, there are different alternatives for multiple testing of slgE to LTPs: FABER, ALEX, and ImmunoCAP ISAC112. The latter contains 9 different LTP: Pru p 3, three nuts LTP (Ara h 9, Cora a 8, Jug r 3), one cereal flour LTP (Tri a 14) and four pollen LTP (Art v 3, Ole e 7, Pla a 3 and Par j 2) and has been widely used in the last years.

The slgE responses to the above mentioned LTP panel could be a reflection of the extension of LTP family recognition and therefore related to severity of the LTP syndrome and the probability of future side reactions to new fruits and vegetables. However, to date there is no validated approach or threshold values for slgE levels. The individual allergens are also available in the ImmunoCAP Specific IgE test.
not need to eliminate rice and maize from his diet. Thus, LTP sensitization to rice and maize was due to cross-reactivity with Pru p3. The message is that the challenge is advisable to avoid unnecessary food elimination.

### Case 2 (unpublished real case)

Female, 52-years old with a 32-year history of peach-peel induced contact urticaria and immediate abdominal pain after peach-juice ingestion. She avoided this fruit since the reaction. The patient tolerated other fruits, vegetables, and tree-nuts. After some time, the patient developed a systemic reaction after eating a complete unpeeled apple (generalized urticaria and angioedema). Soon after she referred oral allergy syndrome with walnut, hazelnut and almond and she avoided other nuts as peanuts for fear of having a new reaction.

The patient had a clinical history of allergic rhinitis to olive pollen since she was 15 years old. SPT to aeroallergens was positive for olive, mugwort, plane-tree pollen and HDM. SPT to plant-food was positive to peach peel, apple, hazelnut, almond and peanut. Specific IgE were positive for Pru p 3 (21.50 kU/L), for walnut (21 kU/L), hazelnut (10.20 kU/L) and peanut Ara h 9 (11.5 kU/L); and negative for peanut Ara h 2. We carried-out a peeled apple (154 g) open food challenge and the patient presented lips angioedema after 70 g of the total dose. She was treated with oral loratadine, and the reaction was resolved.

The patient participated in the clinical study with Pru p 3 SLIT with a commercialized peach extract enriched in Pru p 3 (50 µg/mL) (ALK-Abello S.A.). At the inclusion visit, we performed a DBPCFC with unpeeled peach, (300 mL that is a total dose of 2.5 mg of Pru p 3) and peanut (15 units) The patient presented lips angioedema and OAS after 50 mL of peach juice and oral allergy symptoms with VAS of seven with three consecutive dose of peanut (total of seven fried peanut nuts). The patient was included on a clinical trial and was treated during a year with Pru p 3 SLIT, receiving a 10µg daily dose of Pru p 3. After 12 months of treatment, we observed a mild decrease in the size of papule for peach (10 mm area before Pru p 3-SLIT to 6 mm area after Pru p 3-SLIT) but not to peanut. We observed a decrease in slgE to Pru p 3 (12.3 kU/L) and peanut (6.10 kU/L). During the DBPCFC with peach, the patient tolerated the maximum amount of juice and peanut (15 units).

We recommended to include peach and peanut in a free diet (taking the maximum dose tolerated during DBPCFC). This is a clinical case of a patient suffering from LTP-Syndrome, in which SLIT-Pru p 3 has provided an improvement in tolerance not only for peach but also for other foods related to LTP sensitization such as peanuts. After the study the patient continued with the SLIT for two years and we performed OFC with the other nuts (hazelnut and almond) and unpeel-apple, that were introduced in the diet. The patient refused the oral food challenge with walnut.

### Case 3 (unpublished real case)

Male, 30-year with a 24-years old history of peach-peel induced contact urticaria. He avoided this fruit since the reaction. The patient tolerated other fruits, vegetables, and tree-nuts. Ten years after first symptoms, the patient developed a systemic reaction after ingestion of walnut, hazelnut, and peanut (hives, angioedema, shortness of breath, dizziness). Since the reaction with these nuts, the patient avoided all types of tree-nuts, including pistachio and cashew. SPT to aeroallergens was positive to mugwort and plane-tree pollen. SPT to plant-food was positive to peach peel, hazelnut, and peanut; and negative to almond, pistachio, and cashew. Specific IgE were positive for Pru p 3 (20.6 kU/L), moderate for walnut (4.93 kU/L), hazelnut (0.54 kU/L) and peanut Ara h 9 (15 kU/L); and negative for peanut Ara h 2. Almond Open Food Challenge was positive with a total dose of 5 units of fried almond, presenting the patients oral allergy syndrome and lip angioedema that was treated with IM dexchlorpheniramine. Pistachio and cashew OFCs were negative. Upon DBPCFC with unpeeled-peach and peanut, the patient presented OAS and abdominal pain at intermediate peach dose as well as lips angioedema, erythema, and pruritus after 5 units of fried peanut. The patient was included on clinical study and was treated during a year with Pru p 3-SLIT (as in case 2). After 12 months of treatment, we did not observe a decrease in the peach or peanut wheal size, but we observed a decrease in the levels of slgE to Pru p 3 (10.5 kU/L); and peanut (4.3 kU/L). During the DBPCFC with peach the patient tolerated all the amount of juice and peanut (15 units). This is a clinical case of a patient that developed an LTP syndrome, in which Pru p 3-SLIT induced an improvement in tolerance not only for peach but also of other foods related to LTP sensitization such as peanuts. Currently the patient tolerates other nuts as walnut and hazelnut.

## 6 – Tools

CRD by SPT is possible by using peach extracts highly enriched on Pru p 3, (with very low content of other allergens).<sup>1453</sup> As most of the patients with nsLTP-mediated food allergic reactions will be positive to Pru p 3, this extract should be used in a general screening for inhaled as well as food allergy. Complex nsLTP syndrome patients will eventually react to multiple members of the family. Today, there are different alternatives for multiple testing of slgE to LTPs: FABER, ALEX, and ImmunoCAP ISAC112. The latter contains 9 different LTP: Pru p 3, three nuts LTP (Ara h 9, Cora a 8, Jug r 3), one cereal flour LTP (Tri a 14) and four pollen LTP (Art v 3, Ole e 7, Pla a 3 and Par j 2) and has been widely used in the last years.

The slgE responses to the above mentioned LTP panel could be a reflection of the extension of LTP family recognition and therefore related to severity of the LTP syndrome and the probability of future side reactions to new fruits and vegetables. However, to date there is no validated approach or threshold values for slgE levels. The individual allergens are also available in the ImmunoCAP Specific IgE test.
# C04 – Serum albumins

## Maksymilian Chruszcz, Martine Morisset, Christiane Hilger

### Highlights

* Highly conserved sequences with high amino acid sequence identity

* Minor respiratory allergen of animal dander

* Food allergen of milk and meat

* Allergen implicated in pork-cat and bird-egg syndrome

## 1 – The protein

Bos d 6, Bovine serum albumin (BSA), is a well-characterized protein (Table 82).¹⁴⁶⁵ It is synthesized in the liver and is a major component of plasma. BSA is widely used in biochemical and immunological assays as well as in vaccines, surgical adhesives, hemostatic tissues, and it is a common cell culture ingredient.

The protein architecture of Bos d 6 was resolved in 2012¹⁴⁶⁵ and revealed an α-helical structure composed of three structurally similar domains arranged into a heart-shaped form (Figure 154). The molecule is stabilized by seventeen disulfide bonds. Bos d 6 is denatured by heating to temperatures above 50°C. Helices are partially disrupted and heat-induced aggregation takes place at 60°C.¹⁴⁶⁶

Bos d 6 is a respiratory and food allergen as it is present in bovine dander, milk, and meat. BSA is classified as minor allergen in animal dander, but it is an important meat and milk allergen in the case of uncooked food ingestion. Since Bos d 6 is a thermolabile protein, well-done meat and boiled milk are safe for allergic patients (for details, see chapters on Allergy to furry animals, Allergy to meat, and Allergy to milk).⁴⁷⁷ There is currently no assay available for quantification of Bos d 6 in the environment.

### Family characteristics

- Common tertiary α-helical structure

- Highly conserved sequences with high amino acid sequence identity

- Large secreted molecules of 65 - 69 kDa

- Bind many small molecular compounds

- Thermolabile

- Present in dander, secretions, and meat

## 2 – The family

Serum albumins are large globular proteins synthesized in the liver.¹⁴⁶⁷ They are abundant in plasma and contribute to the

<table><caption>TABLE 82 Characteristics of the prototype protein</caption><thead><tr><th colspan="2">Protein characteristics</th></tr></thead><tbody><tr><td>Allergen source</td><td>Bos domesticus, domestic cattle</td></tr><tr><td>Protein family</td><td>Serum albumin</td></tr><tr><td>UniProtKB accession</td><td>P02769</td></tr><tr><td>Crystal structure</td><td>Yes</td></tr><tr><td>Molecular structure</td><td>Mainly helical</td></tr><tr><td>Theoretical molecular weight</td><td>66.56 kDa</td></tr><tr><td>Length</td><td>607 amino acids; mature protein: 583 amino acids</td></tr><tr><td>Ligand binding</td><td>Yes</td></tr><tr><td>Dimerization</td><td>No</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>17</td></tr><tr><td>Isoelectric point</td><td>5.6</td></tr><tr><td>Synthesis</td><td>Liver, secreted</td></tr><tr><td>Distribution</td><td>Plasma, dander, saliva, milk, meat</td></tr></tbody></table>

<figure><img src="image_277.png" /><figcaption>FIGURE 154 Ribbon model of the three-dimensional structure of Bos d 6 (PDB code: 3V03). N-terminal end in blue, C-terminal end in red.</figcaption></figure>

regulation of colloid osmotic pressure. Serum albumins transport a multitude of metabolites, nutrients, drugs, and other molecules.⁴⁶⁹ They have an α-helical structure with three domains stabilized by several disulfide bridges. Serum albumins change their conformation in order to bind diverse ligands. These proteins present in dander, saliva, milk, and meat are thermolabile and easily denatured in food by cooking. Chicken serum albumin, formerly known as α-livetin, is an allergen of egg yolk. Seven serum albumin allergens are officially recognized by the IUIS Allergen Nomenclature Subcommittee: Bos d 6 (bovine), Can f 3 (dog), Cav p 4 (guinea pig), Equ c 3 (horse), Fel
# C04 – Serum albumins

## Maksymilian Chruszcz, Martine Morisset, Christiane Hilger

### Highlights

* Highly conserved sequences with high amino acid sequence identity

* Minor respiratory allergen of animal dander

* Food allergen of milk and meat

* Allergen implicated in pork-cat and bird-egg syndrome

## 1 – The protein

Bos d 6, Bovine serum albumin (BSA), is a well-characterized protein (Table 82).¹⁴⁶⁵ It is synthesized in the liver and is a major component of plasma. BSA is widely used in biochemical and immunological assays as well as in vaccines, surgical adhesives, hemostatic tissues, and it is a common cell culture ingredient.

The protein architecture of Bos d 6 was resolved in 2012¹⁴⁶⁵ and revealed an α-helical structure composed of three structurally similar domains arranged into a heart-shaped form (Figure 154). The molecule is stabilized by seventeen disulfide bonds. Bos d 6 is denatured by heating to temperatures above 50°C. Helices are partially disrupted and heat-induced aggregation takes place at 60°C.¹⁴⁶⁶

Bos d 6 is a respiratory and food allergen as it is present in bovine dander, milk, and meat. BSA is classified as minor allergen in animal dander, but it is an important meat and milk allergen in the case of uncooked food ingestion. Since Bos d 6 is a thermolabile protein, well-done meat and boiled milk are safe for allergic patients (for details, see chapters on Allergy to furry animals, Allergy to meat, and Allergy to milk).⁴⁷⁷ There is currently no assay available for quantification of Bos d 6 in the environment.

### Family characteristics

- Common tertiary α-helical structure

- Highly conserved sequences with high amino acid sequence identity

- Large secreted molecules of 65 - 69 kDa

- Bind many small molecular compounds

- Thermolabile

- Present in dander, secretions, and meat

## 2 – The family

Serum albumins are large globular proteins synthesized in the liver.¹⁴⁶⁷ They are abundant in plasma and contribute to the

<table><caption>TABLE 82 Characteristics of the prototype protein</caption><thead><tr><th colspan="2">Protein characteristics</th></tr></thead><tbody><tr><td>Allergen source</td><td>Bos domesticus, domestic cattle</td></tr><tr><td>Protein family</td><td>Serum albumin</td></tr><tr><td>UniProtKB accession</td><td>P02769</td></tr><tr><td>Crystal structure</td><td>Yes</td></tr><tr><td>Molecular structure</td><td>Mainly helical</td></tr><tr><td>Theoretical molecular weight</td><td>66.56 kDa</td></tr><tr><td>Length</td><td>607 amino acids; mature protein: 583 amino acids</td></tr><tr><td>Ligand binding</td><td>Yes</td></tr><tr><td>Dimerization</td><td>No</td></tr><tr><td>Glycosylation</td><td>No</td></tr><tr><td>Disulfide bonds</td><td>17</td></tr><tr><td>Isoelectric point</td><td>5.6</td></tr><tr><td>Synthesis</td><td>Liver, secreted</td></tr><tr><td>Distribution</td><td>Plasma, dander, saliva, milk, meat</td></tr></tbody></table>

<figure><img src="image_283.png" /><figcaption>FIGURE 154 Ribbon model of the three-dimensional structure of Bos d 6 (PDB code: 3V03). N-terminal end in blue, C-terminal end in red.</figcaption></figure>

regulation of colloid osmotic pressure. Serum albumins transport a multitude of metabolites, nutrients, drugs, and other molecules.⁴⁶⁹ They have an α-helical structure with three domains stabilized by several disulfide bridges. Serum albumins change their conformation in order to bind diverse ligands. These proteins present in dander, saliva, milk, and meat are thermolabile and easily denatured in food by cooking. Chicken serum albumin, formerly known as α-livetin, is an allergen of egg yolk. Seven serum albumin allergens are officially recognized by the IUIS Allergen Nomenclature Subcommittee: Bos d 6 (bovine), Can f 3 (dog), Cav p 4 (guinea pig), Equ c 3 (horse), Fel
d 2 (cat), Gal d 5 (chicken), and Sus s 1 (pig) (Table 83). However, a number of serum albumins from different animals have been shown to bind IgE and to be cross-reactive: sheep, goat, rabbit, hamster, mouse, rat, and other mammals, as well as birds like pigeon, although these serum albumins are not yet categorized as allergens.¹⁶⁶⁸

Serum albumins have a molecular weight of 65-69 kDa. They are composed of 607-608 amino acids and the signal peptide and a pro-peptide of 18 and four amino acids respectively are cleaved off during maturation. Mammalian serum albumins have highly conserved amino acid sequences¹⁴⁶⁷ and show sequence identities of 72-82% with human serum albumin (HSA). Avian serum albumins display lower identities (46-49%) with HSA and other mammalian serum albumins (42-48%).

Table 84 displays two-by-two comparisons of amino acid identities between HSA and different serum albumins. The identity between HSA and mammalian serum albumins is very high. It was generally assumed that proteins with a sequence identity to a human homologue above 62% were rarely allergenic.¹⁴⁶⁸ Serum albumins constitute a remarkable exception to this rule. Other important animal allergen families such as tropomyosins, β-parvalbumins, and caseins lie below this threshold. IgE-cross-reactivity between mammalian serum albumins has been well documented.¹⁶⁶⁷ All pairs with a high sequence identity (>70%) are potentially cross-reactive. It has been postulated that below 50%, cross-reactivity is rare.¹³,¹⁵²⁸ Cross-reactivity between mammalian and the less-conserved avian serum albumins seems to be rare, but it has been documented in single case reports.¹⁶⁷⁰ Molecules displaying a low level of overall sequence identity may nevertheless share single epitopes composed of

short stretches of sequence identity that lead to patient-dependent IgE-cross-reactivity.

A comparison of the surfaces of some important allergenic mammalian serum albumins visualizes potential cross-reactive B cell epitopes (Figure 155). B- and T-cell recognizing HSA epitopes will be deleted during the immunological education process. Figure 156 illustrates cross-reactivity among some important serum albumins recognized by the IUIS.

## 3 – Clinical relevance

Serum albumins are respiratory allergens present in animal dander and fluids such as milk, serum, urine, and saliva. They are considered minor allergens.⁴⁷⁷ Depending on the study population, approximately 14-50% of cat and dog-allergic patients present IgE to cat or dog albumins. Monosensitization to cat or dog serum albumin seems to be extremely rare in a primary sensitization. IgE to Fel d 2 is usually detected along with IgE directed to a major allergen (such as Fel d 1). On the contrary, the presence of IgE to Can f 3 without any detectable IgE to other dog allergens (Can f 1 or Can f 2) is thought to be a marker of cross-reactivity and the primary sensitization source should be sought after. The clinical relevance of IgE to serum albumins concerning respiratory symptoms, is difficult to explore as IgE to other allergens from the same animal are always present in the same patient. It is generally assumed that they are of low relevance. However, two cases of occupational asthma triggered by inhalation of Bos d 6 have been reported in laboratory workers.¹³⁷⁸,¹⁶⁶⁸ High

<table><caption>TABLE 83 Bos d 6-homologous allergens from animals</caption><thead><tr><td>Animal family</td><td>Allergen source</td><td>Allergen</td></tr></thead><tbody><tr><td>Bovidae</td><td>Domestic cattle (Bos domesticus)</td><td>Bos d 6</td></tr><tr><td>Canidae</td><td>Dog (Canis familiaris)</td><td>Can f 3</td></tr><tr><td>Cavidae</td><td>Guinea pig (Cavia porcellus)</td><td>Cav p 4</td></tr><tr><td>Equidae</td><td>Domestic horse (Equus caballus)</td><td>Equ c 1</td></tr><tr><td>Felidae</td><td>Cat (Felis domesticus)</td><td>Fel d 2</td></tr><tr><td>Phasianidae</td><td>Chicken (Gallus domesticus)</td><td>Gal d 5</td></tr><tr><td>Suidae</td><td>Domestic pig (Sus scrofa)</td><td>Sus s 1</td></tr></tbody></table>

<figure><img src="image_278.png" /><figcaption>TABLE 84 Amino acid identities (%) between allergenic serum albumins and HSA¹⁴⁶⁷. Dark blue shaded areas indicate sequence identities >80%, light shaded areas display identities between 71 and 80%. Figures in bold blue indicate documented IgE-cross-reactivity between albumins. HSA, human serum albumin.</figcaption></figure>
d 2 (cat), Gal d 5 (chicken), and Sus s 1 (pig) (Table 83). However, a number of serum albumins from different animals have been shown to bind IgE and to be cross-reactive: sheep, goat, rabbit, hamster, mouse, rat, and other mammals, as well as birds like pigeon, although these serum albumins are not yet categorized as allergens.¹⁶⁶⁸

Serum albumins have a molecular weight of 65-69 kDa. They are composed of 607-608 amino acids and the signal peptide and a pro-peptide of 18 and four amino acids respectively are cleaved off during maturation. Mammalian serum albumins have highly conserved amino acid sequences¹⁴⁶⁷ and show sequence identities of 72-82% with human serum albumin (HSA). Avian serum albumins display lower identities (46-49%) with HSA and other mammalian serum albumins (42-48%).

Table 84 displays two-by-two comparisons of amino acid identities between HSA and different serum albumins. The identity between HSA and mammalian serum albumins is very high. It was generally assumed that proteins with a sequence identity to a human homologue above 62% were rarely allergenic.¹⁴⁶⁸ Serum albumins constitute a remarkable exception to this rule. Other important animal allergen families such as tropomyosins, β-parvalbumins, and caseins lie below this threshold. IgE-cross-reactivity between mammalian serum albumins has been well documented.¹⁶⁶⁷ All pairs with a high sequence identity (>70%) are potentially cross-reactive. It has been postulated that below 50%, cross-reactivity is rare.¹³,¹⁵²⁸ Cross-reactivity between mammalian and the less-conserved avian serum albumins seems to be rare, but it has been documented in single case reports.¹⁶⁷⁰ Molecules displaying a low level of overall sequence identity may nevertheless share single epitopes composed of

short stretches of sequence identity that lead to patient-dependent IgE-cross-reactivity.

A comparison of the surfaces of some important allergenic mammalian serum albumins visualizes potential cross-reactive B cell epitopes (Figure 155). B- and T-cell recognizing HSA epitopes will be deleted during the immunological education process. Figure 156 illustrates cross-reactivity among some important serum albumins recognized by the IUIS.

### 3 – Clinical relevance

Serum albumins are respiratory allergens present in animal dander and fluids such as milk, serum, urine, and saliva. They are considered minor allergens.⁴⁷⁷ Depending on the study population, approximately 14-50% of cat and dog-allergic patients present IgE to cat or dog albumins. Monosensitization to cat or dog serum albumin seems to be extremely rare in a primary sensitization. IgE to Fel d 2 is usually detected along with IgE directed to a major allergen (such as Fel d 1). On the contrary, the presence of IgE to Can f 3 without any detectable IgE to other dog allergens (Can f 1 or Can f 2) is thought to be a marker of cross-reactivity and the primary sensitization source should be sought after. The clinical relevance of IgE to serum albumins concerning respiratory symptoms, is difficult to explore as IgE to other allergens from the same animal are always present in the same patient. It is generally assumed that they are of low relevance. However, two cases of occupational asthma triggered by inhalation of Bos d 6 have been reported in laboratory workers.¹³⁷⁸,¹⁶⁶⁸ High

<table><caption>TABLE 83 Bos d 6-homologous allergens from animals</caption><thead><tr><td>Animal family</td><td>Allergen source</td><td>Allergen</td></tr></thead><tbody><tr><td>Bovidae</td><td>Domestic cattle (Bos domesticus)</td><td>Bos d 6</td></tr><tr><td>Canidae</td><td>Dog (Canis familiaris)</td><td>Can f 3</td></tr><tr><td>Cavidae</td><td>Guinea pig (Cavia porcellus)</td><td>Cav p 4</td></tr><tr><td>Equidae</td><td>Domestic horse (Equus caballus)</td><td>Equ c 1</td></tr><tr><td>Felidae</td><td>Cat (Felis domesticus)</td><td>Fel d 2</td></tr><tr><td>Phasianidae</td><td>Chicken (Gallus domesticus)</td><td>Gal d 5</td></tr><tr><td>Suidae</td><td>Domestic pig (Sus scrofa)</td><td>Sus s 1</td></tr></tbody></table>

<figure><img src="image_284.png" /><figcaption>TABLE 84 Amino acid identities (%) between allergenic serum albumins and HSA¹⁴⁶⁷. Dark blue shaded areas indicate sequence identities >80%, light shaded areas display identities between 71 and 80%. Figures in bold blue indicate documented IgE-cross-reactivity between albumins. HSA, human serum albumin.</figcaption></figure>
<figure><img src="image_279.png" /><figcaption>**FIGURE 155** Surface representation of BSA molecule (PDB code: 3V03) shown in four side views. Residues that are identical in bovine (Bos d 6), feline (Fel d 2), and porcine (Sus s 1) albumins are colored in blue. The identical residues form large surface patches that can be recognized by cross-reactive antibodies. Residues that are different between Bos d 6, Fel d 2, and Sus s 1 are marked in grey.</figcaption></figure>

<figure><img src="image_280.png" /><figcaption>**FIGURE 156** Cross-reactivities among allergenic serum albumins. All mammalian serum albumins are potentially IgE-cross-reactive. Clinical cross-reactivity between mammalian Fel d 2 and Sus s 1 and avian Gal d 5 are rare and have been documented only from mammal to bird. Solid lines represent documented IgE-cross-reactivity, and dashed lines show hypothetical cross-reactivity.</figcaption></figure>

levels of IgE antibodies to Fel d 2 have been associated with atopic dermatitis in cat-allergic children.⁸⁰⁹

In contrast to its role as a respiratory allergen, serum albumin in food has been shown to elicit minor, moderate, and severe clinical symptoms. Bos d 6 is a component of the milk whey fraction and constitutes about 1% of the total protein content of milk (0.1–0.4 g/L). In a series of 60 children with immediate reactions to milk confirmed by DBPCFC, 61.3% had allergen-specific IgE antibodies to Bos d 6.⁹⁵³ Boiling milk for 10 minutes eliminated skin prick test

responses in subjects reactive to BSA. Cross-reactivity has been described between different mammalian milks. Bos d 6, and the major bovine milk allergens casein and β-lactoglobulin have homologues in milk from other species. Serum albumins are also an important allergen in meat. A high percentage of milk-allergic children (13-20%) are also allergic to beef.⁹⁵³,⁹⁷⁴ In a series of 28 children diagnosed with beef allergy, 92.9% were diagnosed as allergic to milk by skin prick test and DBPCFC. In children with beef allergy, sensitization to Bos d 6 is a marker of cow's milk allergy.⁹⁵³,⁹⁷⁴ Because
<figure><img src="image_285.png" /><figcaption>FIGURE 155 Surface representation of BSA molecule (PDB code: 3V03) shown in four side views. Residues that are identical in bovine (Bos d 6), feline (Fel d 2), and porcine (Sus s 1) albumins are colored in blue. The identical residues form large surface patches that can be recognized by cross-reactive antibodies. Residues that are different between Bos d 6, Fel d 2, and Sus s 1 are marked in grey.</figcaption></figure>

<figure><img src="image_286.png" /><figcaption>FIGURE 156 Cross-reactivities among allergenic serum albumins. All mammalian serum albumins are potentially IgE-cross-reactive. Clinical cross-reactivity between mammalian Fel d 2 and Sus s 1 and avian Gal d 5 are rare and have been documented only from mammal to bird. Solid lines represent documented IgE-cross-reactivity, and dashed lines show hypothetical cross-reactivity.</figcaption></figure>

levels of IgE antibodies to Fel d 2 have been associated with atopic dermatitis in cat-allergic children.<sup>809</sup>

In contrast to its role as a respiratory allergen, serum albumin in food has been shown to elicit minor, moderate, and severe clinical symptoms. Bos d 6 is a component of the milk whey fraction and constitutes about 1% of the total protein content of milk (0.1–0.4 g/L). In a series of 60 children with immediate reactions to milk confirmed by DBPCFC, 61.3% had allergen-specific IgE antibodies to Bos d 6.<sup>953</sup> Boiling milk for 10 minutes eliminated skin prick test

responses in subjects reactive to BSA. Cross-reactivity has been described between different mammalian milks. Bos d 6, and the major bovine milk allergens casein and β-lactoglobulin have homologues in milk from other species. Serum albumins are also an important allergen in meat. A high percentage of milk-allergic children (13-20%) are also allergic to beef.<sup>953,974</sup> In a series of 28 children diagnosed with beef allergy, 92.9% were diagnosed as allergic to milk by skin prick test and DBPCFC. In children with beef allergy, sensitization to Bos d 6 is a marker of cow's milk allergy.<sup>953,974</sup> Because
Bos d 6 is a thermolabile allergen, well-done meat is tolerated by most patients.

Albumins are also involved in cross-reactivity between animal dander and milk or meat. A patient allergic to horse developed a systemic reaction upon ingestion of mare's milk.¹⁴⁷¹ The association between allergy to cat dander and pork meat, known as pork-cat syndrome, was described more than a decade ago in Europe.¹⁴⁷⁰ Among two groups of cat-allergic patients, 14 and 23% had specific IgE to Fel d 2 and 3-10% had cross-reacting IgE to porcine albumin.⁷⁹⁵ Among those, one of three experienced clinical symptoms upon ingestion of pork meat. Thus, 1-3% of cat-allergic patients could develop pork-cat syndrome. This syndrome has also been described in the United States and should be differentiated from the delayed meat allergy based on IgE to alpha-gal (see chapter B14).¹⁰⁹³ Although pork-cat syndrome represents the most frequent clinical cross-reactivity between mammalian dander and meat, additional isolated cases have been described, such as between horse meat and animal dander or between pork and horse dander.¹⁴⁷¹ However, pork-cat syndrome may not be limited to food, as shown by a case of occupational asthma triggered by handling of cured meat.¹⁴⁷²

Chicken serum albumin (Gal d 5) is an inhalant and food allergen implicated in the bird-egg syndrome¹⁴⁷³ or egg-bird syndrome, depending on the primary exposure.¹⁶⁷⁰ It is present in egg yolk and it has also been detected in domestic air samples. Bronchial challenges elicited asthmatic responses in six asthmatic patients. IgE reactivity was reduced to 88% after heating at 90°C for 30 minutes. Chicken serum albumin Gal d 5 does not share any sequence identity with ovalbumin Gal d 2, a storage protein and allergen from egg white.

The wide use of BSA in cell culture media holds new risks. Several case reports have shown that Bos d 6, an ingredient of the culture medium of spermatozooids, has provoked severe anaphylactic reactions upon artificial insemination.⁴⁷⁷ Presence of BSA in media during the production of vaccines is another potential risk factor. Therefore, WHO has set a guideline stating that no more than 50 ng of BSA can be present in a single vaccine dose, likely lowering the number of reactions to BSA in vaccines.¹⁴⁷⁴ However, some cases of allergic reactions to vaccines are most likely associated with continued presence of BSA.¹⁴⁷⁵ Recently, BioGlue, a surgical adhesive composed of BSA, has been implicated in two cases of perioperative anaphylaxis in patients allergic to cat and sensitized to Fel d 2.¹⁴⁷⁶,¹⁴⁷⁷ The high concentration of BSA and a brief exposure of cross-reactive epitopes before complete denaturation and strong protein cross-linking may contribute to the severity of the reaction.

Allergy to HSA is very rare even in cases when recombinant protein is used.¹⁴⁷⁸ However, recently there were two reports indicating that HSA is responsible for anaphylactic reactions.¹⁴⁷⁹,¹⁴⁸⁰ In one of these cases, the authors speculated that reaction to HSA may be associated with the presence of small molecular compounds that are used to prevent aggregation of commercial formulations of HSA or used during sterilization, or that are present in tubing used

to administer the albumin solution.¹⁴⁸⁰ Other studies suggested that modification of HSA by isocyanates, which are used in production of polyurethane, may lead to formation of new antigens that can cause asthma.¹⁴⁸¹,¹⁴⁸²

## Clinical relevance

* Minor respiratory allergen from animal dander

* Mean sensitization rates of up to 30% in patients allergic to furry animals

* Allergen implicated in pork-cat syndrome

* Meat and milk allergen

* May elicit severe symptoms upon ingestion of uncooked or boiled food

* Allergen implicated in bird-egg syndromeNo molecule-based therapeutic approach available

As serum albumins are minor allergens, there is no current research on the development of hypoallergenic molecules. Current immunotherapies available contain animal dander extracts and may vary in albumin content.

## 4 – Clinical management

A careful record of the clinical history such as the presence of pets at home or regular pet contact is of great value. Skin prick test or allergen-specific IgE using animal dander will confirm animal sensitization. In order to define the primary sensitization source, specific IgE to major allergens such as Fel d 1, Can f 1, Can f 2, or Can f 5 should be determined (see chapter B06). The number of available components is increasing and hopefully more allergens including those of small furry pets will be available on all commercial platforms. IgE antibodies directed to serum albumins are a marker of cross-reactivity but do not mandatorily imply clinical cross-reactivity. Patients with IgE to serum albumins should be advised to avoid mammalian pets as they may experience clinical symptoms upon contact with any pet. As mammalian serum albumins are highly cross-reactive, the choice of serum albumins included in the determination of allergen-specific IgE should be guided by clinical history (Figures 157-159). Specific IgE are mostly positive to Can f 3 and Fel d 2, whereas IgE reactivity against Bos d 6 and Sus s 1 are less frequent. Gal d 5 should be considered as an independent allergen, as homology to mammalian albumins is very low. Patients with moderate to high levels of IgE to serum albumins are at risk to develop symptoms upon ingestion of raw milk and raw or medium cooked meat such as sausages, ham, and steaks. Levels of IgE to Bos d 6 and Sus s 1 should be determined and patients should be carefully advised. As serum albumins
Bos d 6 is a thermolabile allergen, well-done meat is tolerated by most patients.

Albumins are also involved in cross-reactivity between animal dander and milk or meat. A patient allergic to horse developed a systemic reaction upon ingestion of mare's milk.¹⁴⁷¹ The association between allergy to cat dander and pork meat, known as pork-cat syndrome, was described more than a decade ago in Europe.¹⁴⁷⁰ Among two groups of cat-allergic patients, 14 and 23% had specific IgE to Fel d 2 and 3-10% had cross-reacting IgE to porcine albumin.⁷⁹⁵ Among those, one of three experienced clinical symptoms upon ingestion of pork meat. Thus, 1-3% of cat-allergic patients could develop pork-cat syndrome. This syndrome has also been described in the United States and should be differentiated from the delayed meat allergy based on IgE to alpha-gal (see chapter B14).¹⁰⁹³ Although pork-cat syndrome represents the most frequent clinical cross-reactivity between mammalian dander and meat, additional isolated cases have been described, such as between horse meat and animal dander or between pork and horse dander.¹⁴⁷¹ However, pork-cat syndrome may not be limited to food, as shown by a case of occupational asthma triggered by handling of cured meat.¹⁴⁷²

Chicken serum albumin (Gal d 5) is an inhalant and food allergen implicated in the bird-egg syndrome¹⁴⁷³ or egg-bird syndrome, depending on the primary exposure.¹⁶⁷⁰ It is present in egg yolk and it has also been detected in domestic air samples. Bronchial challenges elicited asthmatic responses in six asthmatic patients. IgE reactivity was reduced to 88% after heating at 90°C for 30 minutes. Chicken serum albumin Gal d 5 does not share any sequence identity with ovalbumin Gal d 2, a storage protein and allergen from egg white.

The wide use of BSA in cell culture media holds new risks. Several case reports have shown that Bos d 6, an ingredient of the culture medium of spermatozooids, has provoked severe anaphylactic reactions upon artificial insemination.⁴⁷⁷ Presence of BSA in media during the production of vaccines is another potential risk factor. Therefore, WHO has set a guideline stating that no more than 50 ng of BSA can be present in a single vaccine dose, likely lowering the number of reactions to BSA in vaccines.¹⁴⁷⁴ However, some cases of allergic reactions to vaccines are most likely associated with continued presence of BSA.¹⁴⁷⁵ Recently, BioGlue, a surgical adhesive composed of BSA, has been implicated in two cases of perioperative anaphylaxis in patients allergic to cat and sensitized to Fel d 2.¹⁴⁷⁶,¹⁴⁷⁷ The high concentration of BSA and a brief exposure of cross-reactive epitopes before complete denaturation and strong protein cross-linking may contribute to the severity of the reaction.

Allergy to HSA is very rare even in cases when recombinant protein is used.¹⁴⁷⁸ However, recently there were two reports indicating that HSA is responsible for anaphylactic reactions.¹⁴⁷⁹,¹⁴⁸⁰ In one of these cases, the authors speculated that reaction to HSA may be associated with the presence of small molecular compounds that are used to prevent aggregation of commercial formulations of HSA or used during sterilization, or that are present in tubing used

to administer the albumin solution.¹⁴⁸⁰ Other studies suggested that modification of HSA by isocyanates, which are used in production of polyurethane, may lead to formation of new antigens that can cause asthma.¹⁴⁸¹,¹⁴⁸²

## Clinical relevance

* Minor respiratory allergen from animal dander

* Mean sensitization rates of up to 30% in patients allergic to furry animals

* Allergen implicated in pork-cat syndrome

* Meat and milk allergen

* May elicit severe symptoms upon ingestion of uncooked or boiled food

* Allergen implicated in bird-egg syndromeNo molecule-based therapeutic approach available

As serum albumins are minor allergens, there is no current research on the development of hypoallergenic molecules. Current immunotherapies available contain animal dander extracts and may vary in albumin content.

## 4 – Clinical management

A careful record of the clinical history such as the presence of pets at home or regular pet contact is of great value. Skin prick test or allergen-specific IgE using animal dander will confirm animal sensitization. In order to define the primary sensitization source, specific IgE to major allergens such as Fel d 1, Can f 1, Can f 2, or Can f 5 should be determined (see chapter B06). The number of available components is increasing and hopefully more allergens including those of small furry pets will be available on all commercial platforms. IgE antibodies directed to serum albumins are a marker of cross-reactivity but do not mandatorily imply clinical cross-reactivity. Patients with IgE to serum albumins should be advised to avoid mammalian pets as they may experience clinical symptoms upon contact with any pet. As mammalian serum albumins are highly cross-reactive, the choice of serum albumins included in the determination of allergen-specific IgE should be guided by clinical history (Figures 157-159). Specific IgE are mostly positive to Can f 3 and Fel d 2, whereas IgE reactivity against Bos d 6 and Sus s 1 are less frequent. Gal d 5 should be considered as an independent allergen, as homology to mammalian albumins is very low. Patients with moderate to high levels of IgE to serum albumins are at risk to develop symptoms upon ingestion of raw milk and raw or medium cooked meat such as sausages, ham, and steaks. Levels of IgE to Bos d 6 and Sus s 1 should be determined and patients should be carefully advised. As serum albumins
<figure><img src="image_281.png" /><figcaption>FIGURE 157 Added value of the use of single allergens in the case of a positive IgE test to cat dander</figcaption></figure>

<figure><img src="image_282.png" /><figcaption>FIGURE 158 Added value of the use of single allergens in the case of a positive IgE test to milk</figcaption></figure>

<figure><img src="image_283.png" /><figcaption>FIGURE 159 Added value of the use of single allergens in the case of a positive IgE test to meat</figcaption></figure>

are thermolabile, well-cooked meat and pasteurized or boiled milk
may be tolerated.

5 - Clinical cases

**Case 1** (published<sup>795)</sup>

**Clinical history:** A 17-year-old girl with a history of rhinitis and asthma when exposed to cat experienced anaphylactic reactions on two occasions after ingestion of pork ham or sausage.

**Tests with extracts:** The patient had positive skin prick tests to cat dander and pork. Specific IgE were positive for cat dander (>100 kU/L) and pork (22 kU/L).

**Tests with molecules:** Specific IgE was positive to several animal serum albumins Fel d 2: (165 kU/L), Can f 3: (37 kU/L), Sus s 1 (22 kU/L) and Bos d 6 (2.5 kU/L). IgE to Sus s 1 could be totally inhibited by prior incubation of the patient's serum with Fel d 2, confirming a primary sensitization to cat.

**Conclusion:** Results were consistent with pork-cat syndrome and the patient was advised to avoid eating pork.
<figure><img src="image_287.png" /><figcaption>FIGURE 157 Added value of the use of single allergens in the case of a positive IgE test to cat dander</figcaption></figure>

<figure><img src="image_288.png" /><figcaption>FIGURE 158 Added value of the use of single allergens in the case of a positive IgE test to milk</figcaption></figure>

<figure><img src="image_289.png" /><figcaption>FIGURE 159 Added value of the use of single allergens in the case of a positive IgE test to meat</figcaption></figure>

are thermolabile, well-cooked meat and pasteurized or boiled milk
may be tolerated.

5 - Clinical cases

**Case 1** (published⁷⁹)

**Clinical history:** A 17-year-old girl with a history of rhinitis and asthma when exposed to cat experienced anaphylactic reactions on two occasions after ingestion of pork ham or sausage.

**Tests with extracts:** The patient had positive skin prick tests to cat dander and pork. Specific IgE were positive for cat dander (>100 kU/L) and pork (22 kU/L).

**Tests with molecules:** Specific IgE was positive to several animal serum albumins Fel d 2: (165 kU/L), Can f 3: (37 kU/L), Sus s 1 (22 kU/L) and Bos d 6 (2.5 kU/L). IgE to Sus s 1 could be totally inhibited by prior incubation of the patient's serum with Fel d 2, confirming a primary sensitization to cat.

**Conclusion:** Results were consistent with pork-cat syndrome and the patient was advised to avoid eating pork.